EcoliWiki - User contributions [en]

Excision Repair by the Uvr System

2012-05-09T17:43:23Z

Mariewong: /* Translocation of UvrD */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
Random mutations were made in the helix-turn-helix motifs of functioning UvrA proteins in order to study these regions. Degenerate oligonucleotide-directed mutagenesis was used to create the random mutations, and from this, 12 single substitution mutants were isolated and failed to show UV resistance in the ''uvrA'' gene-absent strain. Of these mutants, UvrA proteins G502D and V508D were chosen to study further through purification and classification because they carry mutations at the predicted positions of the helix-turn-helix motif. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA, UvrB, and UvrA and UvrB together with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein bound to the column, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to the UvrC column is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. UvrB and UvrC was found to have a lower affinity for each other than the UvrA-UvrB interaction, and it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
ATP is a significant component in the delivery of UvrB to DNA and the incision process which follows. Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. This result suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP in the complexes was hydrolyzed, the experiments were repeated using either [α-32P]ATP or [γ-32P]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured using silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that there was 5- to 10-fold more UvrB than nucleotide on the complexes, indicating that UvrB does not need to bind to ATP in order to bind to DNA.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP slightly inhibited ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
UvrD is a helicase and translocase that functions in excision repair to remove the damaged segment of DNA so that DNA polymerase can fill in the gap. Separation of the UvrD helicase and translocase activities is possible in vitro. Under conditions where UvrD is a monomer, it can move processively along single-stranded DNA, but monomeric UvrB cannot unwind double-stranded DNA.<ref name='PMID:12527298'/>

Tomko et al.<ref name='PMID:22342931'/> have taken advantage of this observation to study the mechanism of UvrD translocation in vitro. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of ''uvrA'' Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both the ''uvrA'' and ''ssb'' genes, and so was used to in an experiment to study ''uvrA'' regulation. Location of ''ssb'' was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating ''uvrA'', thus determining the site as that of ''uvrA'', were used to locate the ''uvrA'' promoter. This fragment was also useful in establishing the nucleotide sequence of the ''uvrA'' promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the ''uvrA'' gene. By specificially binding to the promoter-operator region of ''uvrA'' , LexA guards the ''uvrA'' promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the ''recA'', ''lexA'', and ''uvrB'' genes. It was shown that ''uvrA''’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-09T17:42:25Z

Mariewong: /* Translocation of UvrD */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
Random mutations were made in the helix-turn-helix motifs of functioning UvrA proteins in order to study these regions. Degenerate oligonucleotide-directed mutagenesis was used to create the random mutations, and from this, 12 single substitution mutants were isolated and failed to show UV resistance in the ''uvrA'' gene-absent strain. Of these mutants, UvrA proteins G502D and V508D were chosen to study further through purification and classification because they carry mutations at the predicted positions of the helix-turn-helix motif. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA, UvrB, and UvrA and UvrB together with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein bound to the column, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to the UvrC column is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. UvrB and UvrC was found to have a lower affinity for each other than the UvrA-UvrB interaction, and it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
ATP is a significant component in the delivery of UvrB to DNA and the incision process which follows. Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. This result suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP in the complexes was hydrolyzed, the experiments were repeated using either [α-32P]ATP or [γ-32P]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured using silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that there was 5- to 10-fold more UvrB than nucleotide on the complexes, indicating that UvrB does not need to bind to ATP in order to bind to DNA.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP slightly inhibited ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
UvrD is a helicase and translocase that functions in excision repair to remove the damaged segment of DNA so that DNA polymerase can fill in the gap. Separation of the helicase and translocase activities of UvrD is possible in vitro. Under conditions where UvrD is a monomer, it can move processively along single-stranded DNA, but monomeric UvrB cannot unwind double-stranded DNA.<ref name='PMID:12527298'/>
Tomko et al.<ref name='PMID:22342931'/> have taken advantage of this observation to study the mechanism of UvrD translocation in vitro. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of ''uvrA'' Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both the ''uvrA'' and ''ssb'' genes, and so was used to in an experiment to study ''uvrA'' regulation. Location of ''ssb'' was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating ''uvrA'', thus determining the site as that of ''uvrA'', were used to locate the ''uvrA'' promoter. This fragment was also useful in establishing the nucleotide sequence of the ''uvrA'' promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the ''uvrA'' gene. By specificially binding to the promoter-operator region of ''uvrA'' , LexA guards the ''uvrA'' promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the ''recA'', ''lexA'', and ''uvrB'' genes. It was shown that ''uvrA''’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-09T16:57:00Z

Mariewong: /* UvrB Delivery to Damaged Sites in DNA by UvrA */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
Random mutations were made in the helix-turn-helix motifs of functioning UvrA proteins in order to study these regions. Degenerate oligonucleotide-directed mutagenesis was used to create the random mutations, and from this, 12 single substitution mutants were isolated and failed to show UV resistance in the ''uvrA'' gene-absent strain. Of these mutants, UvrA proteins G502D and V508D were chosen to study further through purification and classification because they carry mutations at the predicted positions of the helix-turn-helix motif. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA, UvrB, and UvrA and UvrB together with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein bound to the column, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to the UvrC column is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. UvrB and UvrC was found to have a lower affinity for each other than the UvrA-UvrB interaction, and it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
ATP is a significant component in the delivery of UvrB to DNA and the incision process which follows. Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. This result suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP in the complexes was hydrolyzed, the experiments were repeated using either [α-32P]ATP or [γ-32P]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured using silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that there was 5- to 10-fold more UvrB than nucleotide on the complexes, indicating that UvrB does not need to bind to ATP in order to bind to DNA.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP slightly inhibited ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of ''uvrA'' Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both the ''uvrA'' and ''ssb'' genes, and so was used to in an experiment to study ''uvrA'' regulation. Location of ''ssb'' was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating ''uvrA'', thus determining the site as that of ''uvrA'', were used to locate the ''uvrA'' promoter. This fragment was also useful in establishing the nucleotide sequence of the ''uvrA'' promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the ''uvrA'' gene. By specificially binding to the promoter-operator region of ''uvrA'' , LexA guards the ''uvrA'' promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the ''recA'', ''lexA'', and ''uvrB'' genes. It was shown that ''uvrA''’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-09T16:51:39Z

Mariewong: /* DNA Damage Recognition by UvrA */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
Random mutations were made in the helix-turn-helix motifs of functioning UvrA proteins in order to study these regions. Degenerate oligonucleotide-directed mutagenesis was used to create the random mutations, and from this, 12 single substitution mutants were isolated and failed to show UV resistance in the ''uvrA'' gene-absent strain. Of these mutants, UvrA proteins G502D and V508D were chosen to study further through purification and classification because they carry mutations at the predicted positions of the helix-turn-helix motif. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein bound to the column, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to the UvrC column is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. UvrB and UvrC was found to have a lower affinity for each other than the UvrA-UvrB interaction, and it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
ATP is a significant component in the delivery of UvrB to DNA and the incision process which follows. Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. This result suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP in the complexes was hydrolyzed, the experiments were repeated using either [α-32P]ATP or [γ-32P]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured using silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that there was 5- to 10-fold more UvrB than nucleotide on the complexes, indicating that UvrB does not need to bind to ATP in order to bind to DNA.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP slightly inhibited ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of ''uvrA'' Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both the ''uvrA'' and ''ssb'' genes, and so was used to in an experiment to study ''uvrA'' regulation. Location of ''ssb'' was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating ''uvrA'', thus determining the site as that of ''uvrA'', were used to locate the ''uvrA'' promoter. This fragment was also useful in establishing the nucleotide sequence of the ''uvrA'' promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the ''uvrA'' gene. By specificially binding to the promoter-operator region of ''uvrA'' , LexA guards the ''uvrA'' promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the ''recA'', ''lexA'', and ''uvrB'' genes. It was shown that ''uvrA''’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-09T16:51:14Z

Mariewong: /* DNA Damage Recognition by UvrA */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
Random mutations were made in the helix-turn-helix motifs of functioning UvrA proteins in order to study these regions. Degenerate oligonucleotide-directed mutagenesis was used to create these random mutations, and from this, 12 single substitution mutants were isolated and failed to show UV resistance in the ''uvrA'' gene-absent strain. Of these mutants, UvrA proteins G502D and V508D were chosen to study further through purification and classification because they carry mutations at the predicted positions of the helix-turn-helix motif. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein bound to the column, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to the UvrC column is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. UvrB and UvrC was found to have a lower affinity for each other than the UvrA-UvrB interaction, and it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
ATP is a significant component in the delivery of UvrB to DNA and the incision process which follows. Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. This result suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP in the complexes was hydrolyzed, the experiments were repeated using either [α-32P]ATP or [γ-32P]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured using silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that there was 5- to 10-fold more UvrB than nucleotide on the complexes, indicating that UvrB does not need to bind to ATP in order to bind to DNA.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP slightly inhibited ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of ''uvrA'' Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both the ''uvrA'' and ''ssb'' genes, and so was used to in an experiment to study ''uvrA'' regulation. Location of ''ssb'' was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating ''uvrA'', thus determining the site as that of ''uvrA'', were used to locate the ''uvrA'' promoter. This fragment was also useful in establishing the nucleotide sequence of the ''uvrA'' promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the ''uvrA'' gene. By specificially binding to the promoter-operator region of ''uvrA'' , LexA guards the ''uvrA'' promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the ''recA'', ''lexA'', and ''uvrB'' genes. It was shown that ''uvrA''’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-09T14:59:02Z

Mariewong: /* DNA Incision by UvrB and UvrC */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of functioning UvrA proteins. Degenerate oligonucleotide-directed mutagenesis was used to create these random mutations, and from this, 12 single substitution mutants were isolated and failed to show UV resistance in the ''uvrA'' gene-absent strain. Of these mutants, UvrA proteins G502D and V508D were chosen to study further through purification and classification because they carry mutations at the predicted positions of the helix-turn-helix motif. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein bound to the column, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to the UvrC column is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. UvrB and UvrC was found to have a lower affinity for each other than the UvrA-UvrB interaction, and it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
ATP is a significant component in the delivery of UvrB to DNA and the incision process which follows. Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. This result suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP in the complexes was hydrolyzed, the experiments were repeated using either [α-32P]ATP or [γ-32P]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured using silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that there was 5- to 10-fold more UvrB than nucleotide on the complexes, indicating that UvrB does not need to bind to ATP in order to bind to DNA.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP slightly inhibited ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of ''uvrA'' Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both the ''uvrA'' and ''ssb'' genes, and so was used to in an experiment to study ''uvrA'' regulation. Location of ''ssb'' was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating ''uvrA'', thus determining the site as that of ''uvrA'', were used to locate the ''uvrA'' promoter. This fragment was also useful in establishing the nucleotide sequence of the ''uvrA'' promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the ''uvrA'' gene. By specificially binding to the promoter-operator region of ''uvrA'' , LexA guards the ''uvrA'' promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the ''recA'', ''lexA'', and ''uvrB'' genes. It was shown that ''uvrA''’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-09T14:54:28Z

Mariewong: /* DNA Incision by UvrB and UvrC */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of functioning UvrA proteins. Degenerate oligonucleotide-directed mutagenesis was used to create these random mutations, and from this, 12 single substitution mutants were isolated and failed to show UV resistance in the ''uvrA'' gene-absent strain. Of these mutants, UvrA proteins G502D and V508D were chosen to study further through purification and classification because they carry mutations at the predicted positions of the helix-turn-helix motif. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein bound to the column, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to the UvrC column is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. UvrB and UvrC was found to have a lower affinity for each other than the UvrA-UvrB interaction, and it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. This result suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP in the complexes was hydrolyzed, the experiments were repeated using either [α-32P]ATP or [γ-32P]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured using silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that there was 5- to 10-fold more UvrB than nucleotide on the complexes, indicating that UvrB does not need to bind to ATP in order to bind to DNA.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP slightly inhibited ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of ''uvrA'' Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both the ''uvrA'' and ''ssb'' genes, and so was used to in an experiment to study ''uvrA'' regulation. Location of ''ssb'' was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating ''uvrA'', thus determining the site as that of ''uvrA'', were used to locate the ''uvrA'' promoter. This fragment was also useful in establishing the nucleotide sequence of the ''uvrA'' promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the ''uvrA'' gene. By specificially binding to the promoter-operator region of ''uvrA'' , LexA guards the ''uvrA'' promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the ''recA'', ''lexA'', and ''uvrB'' genes. It was shown that ''uvrA''’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-09T14:32:37Z

Mariewong: /* UvrB and UvrC Interaction */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of functioning UvrA proteins. Degenerate oligonucleotide-directed mutagenesis was used to create these random mutations, and from this, 12 single substitution mutants were isolated and failed to show UV resistance in the ''uvrA'' gene-absent strain. Of these mutants, UvrA proteins G502D and V508D were chosen to study further through purification and classification because they carry mutations at the predicted positions of the helix-turn-helix motif. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein bound to the column, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to the UvrC column is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. UvrB and UvrC was found to have a lower affinity for each other than the UvrA-UvrB interaction, and it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. This result suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP in the complexes was hydrolyzed, the experiments were repeated using either [α-32P]ATP or [γ-32P]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that there was 5- to 10-fold more UvrB than nucleotide on the complexes, indicating that UvrB does not need to bind to ATP in order to bind to DNA.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP slightly inhibited ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of ''uvrA'' Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both the ''uvrA'' and ''ssb'' genes, and so was used to in an experiment to study ''uvrA'' regulation. Location of ''ssb'' was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating ''uvrA'', thus determining the site as that of ''uvrA'', were used to locate the ''uvrA'' promoter. This fragment was also useful in establishing the nucleotide sequence of the ''uvrA'' promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the ''uvrA'' gene. By specificially binding to the promoter-operator region of ''uvrA'' , LexA guards the ''uvrA'' promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the ''recA'', ''lexA'', and ''uvrB'' genes. It was shown that ''uvrA''’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-09T14:27:48Z

Mariewong: /* DNA Damage Recognition by UvrA */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of functioning UvrA proteins. Degenerate oligonucleotide-directed mutagenesis was used to create these random mutations, and from this, 12 single substitution mutants were isolated and failed to show UV resistance in the ''uvrA'' gene-absent strain. Of these mutants, UvrA proteins G502D and V508D were chosen to study further through purification and classification because they carry mutations at the predicted positions of the helix-turn-helix motif. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein bound to the column, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to the UvrC column is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. This result suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP in the complexes was hydrolyzed, the experiments were repeated using either [α-32P]ATP or [γ-32P]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that there was 5- to 10-fold more UvrB than nucleotide on the complexes, indicating that UvrB does not need to bind to ATP in order to bind to DNA.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP slightly inhibited ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of ''uvrA'' Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both the ''uvrA'' and ''ssb'' genes, and so was used to in an experiment to study ''uvrA'' regulation. Location of ''ssb'' was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating ''uvrA'', thus determining the site as that of ''uvrA'', were used to locate the ''uvrA'' promoter. This fragment was also useful in establishing the nucleotide sequence of the ''uvrA'' promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the ''uvrA'' gene. By specificially binding to the promoter-operator region of ''uvrA'' , LexA guards the ''uvrA'' promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the ''recA'', ''lexA'', and ''uvrB'' genes. It was shown that ''uvrA''’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-08T20:45:15Z

Mariewong: /* Regulation of uvrA Gene Transcription by LexA Protein */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the predicted positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein bound to the column, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to the UvrC column is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. This result suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP in the complexes was hydrolyzed, the experiments were repeated using either [α-32P]ATP or [γ-32P]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that there was 5- to 10-fold more UvrB than nucleotide on the complexes, indicating that UvrB does not need to bind to ATP in order to bind to DNA.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP slightly inhibited ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of ''uvrA'' Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both the ''uvrA'' and ''ssb'' genes, and so was used to in an experiment to study ''uvrA'' regulation. Location of ''ssb'' was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating ''uvrA'', thus determining the site as that of ''uvrA'', were used to locate the ''uvrA'' promoter. This fragment was also useful in establishing the nucleotide sequence of the ''uvrA'' promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the ''uvrA'' gene. By specificially binding to the promoter-operator region of ''uvrA'' , LexA guards the ''uvrA'' promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the ''recA'', ''lexA'', and ''uvrB'' genes. It was shown that ''uvrA''’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-08T20:44:38Z

Mariewong: /* Regulation of uvrA Gene Transcription by LexA Protein */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the predicted positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein bound to the column, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to the UvrC column is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. This result suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP in the complexes was hydrolyzed, the experiments were repeated using either [α-32P]ATP or [γ-32P]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that there was 5- to 10-fold more UvrB than nucleotide on the complexes, indicating that UvrB does not need to bind to ATP in order to bind to DNA.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP slightly inhibited ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of ''uvrA'' Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both ''uvrA'' and ''ssb'' genes, and so was used to in an experiment to study ''uvrA'' regulation. Location of ''ssb'' was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating ''uvrA'', thus determining the site as that of ''uvrA'', were used to locate the ''uvrA'' promoter. This fragment was also useful in establishing the nucleotide sequence of the ''uvrA'' promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the ''uvrA'' gene. By specificially binding to the promoter-operator region of ''uvrA'' , LexA guards the ''uvrA'' promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the ''recA'', ''lexA'', and ''uvrB'' genes. It was shown that ''uvrA''’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-08T20:39:11Z

Mariewong: /* DNA Incision by UvrB and UvrC */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the predicted positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein bound to the column, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to the UvrC column is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. This result suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP in the complexes was hydrolyzed, the experiments were repeated using either [α-32P]ATP or [γ-32P]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that there was 5- to 10-fold more UvrB than nucleotide on the complexes, indicating that UvrB does not need to bind to ATP in order to bind to DNA.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP slightly inhibited ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of uvrA Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-08T20:28:40Z

Mariewong: /* UvrB and UvrC Interaction */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the predicted positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein bound to the column, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to the UvrC column is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of uvrA Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-08T20:26:50Z

Mariewong: /* UvrB Delivery to Damaged Sites in DNA by UvrA */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the predicted positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The requirements for using UvrB binding to DNA were examined by incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA binds to both UV-irradiated and non-irradiated DNA, while UvrB binds to DNA only when the following conditions were met: the DNA was UV-irradiated, UvrA was present in the reaction mixture, and ATP was present in the column buffer, as well as in the reaction mixture. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of uvrA Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-08T20:23:00Z

Mariewong: /* General Description */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5 bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by a DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the predicted positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and non-irradiated DNA, while UvrB would bind to DNA only when the following conditions are present at once: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of uvrA Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-08T20:20:54Z

Mariewong: /* DNA Damage Recognition by UvrA */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the predicted positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA was then compared with that of the mutant UvrA proteins. DNA binding was assayed using both irradiated and non-irradiated DNA. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and non-irradiated DNA, while UvrB would bind to DNA only when the following conditions are present at once: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of uvrA Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-08T20:11:21Z

Mariewong: /* General Description */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants that lack one or more of the Uvr proteins are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts. These lesions are lethal to the cells because it prevents DNA transcription.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with and 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the presumed positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, where the DNA was either irradiated or non-irradiated. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and non-irradiated DNA, while UvrB would bind to DNA only when the following conditions are present at once: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of uvrA Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-08T16:40:05Z

Mariewong: /* DNA Incision by UvrB and UvrC */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants lack one or more of the Uvr proteins and are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the presumed positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, where the DNA was either irradiated or non-irradiated. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and non-irradiated DNA, while UvrB would bind to DNA only when the following conditions are present at once: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4ºC and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of uvrA Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-08T16:39:46Z

Mariewong: /* DNA Incision by UvrB and UvrC */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants lack one or more of the Uvr proteins and are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the presumed positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, where the DNA was either irradiated or non-irradiated. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and non-irradiated DNA, while UvrB would bind to DNA only when the following conditions are present at once: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4 C and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4ºC. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of uvrA Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-08T16:38:25Z

Mariewong: /* UvrD */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants lack one or more of the Uvr proteins and are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the presumed positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, where the DNA was either irradiated or non-irradiated. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and non-irradiated DNA, while UvrB would bind to DNA only when the following conditions are present at once: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4 C and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4 C. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===Translocation of UvrD===
A UvrD monomer is a single-stranded DNA translocase that is able to processively perform only in vivo. In preparation of the study, an oligodeoxythymidylate at the 5’-end of the ssDNA was labeled with Cy3 or fluorescein. UvrD monomer binding to ssDNA occurs randomly, so after the UvrD:ssDNA complex formed, it was mixed with buffer T20, ATP, MgCl2, and heparin. Once the UvrD monomer reached the Cy3-labeled and fluorescein-labeled 5’-ends of the ssDNA, the fluorescence intensity was enhanced and quenched, respectively. It was observed that at saturating levels of ATP (500 μM), UvrD monomers translocate in the 3’ to 5’ direction, hydrolyzing 1 ATP per DNA base translocated. Also seen was a kinetic step of 4-5 nucleotides per step, suggesting that a pause occurs every 4-5 nucleotides translocated. With a decrease in ATP concentration to the range 10-500 μM, the same procedures were carried out to examine the effects on UvrD monomer translocation along ssDNA. The coupling ATP stoichiometry of about 1 ATP per DNA base translocated remained constant at differing ATP concentrations, however, the translocation kinetic step increased with decreasing ATP concentration, most notably below ATP concentrations of 50 μM; the translocation kinetic step at 50 μM ATP remained at 4-5 nucleotides per step, while at 10 μM ATP, it was about 7 nucleotides per step. The consistency which was observed in ATP coupling despite ATP concentration changes suggests that the uvrD monomer ATP hydrolysis is tightly coupled to the forward translocation of UvrD along ssDNA with minimal occurrences of ineffective ATP hydrolysis, and the increase in the translocation kinetic step due to decreases in ATP concentration indicates that UvrD pauses more frequently during translocation at low ATP, also with little futile ATP hydrolysis.<ref name='PMID:22342931'/>

==Regulation==
===Regulation of uvrA Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-08T16:35:38Z

Mariewong: /* Function */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants lack one or more of the Uvr proteins and are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the presumed positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, where the DNA was either irradiated or non-irradiated. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and non-irradiated DNA, while UvrB would bind to DNA only when the following conditions are present at once: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4 C and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4 C. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===UvrD===

==Regulation==
===Regulation of uvrA Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-08T16:35:18Z

Mariewong: /* Function */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants lack one or more of the Uvr proteins and are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the presumed positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, where the DNA was either irradiated or non-irradiated. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and non-irradiated DNA, while UvrB would bind to DNA only when the following conditions are present at once: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4 C and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4 C. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

===UvrD===

==Regulation==
===Regulation of uvrA Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-07T19:06:16Z

Mariewong: /* Regulation Through Inhibition of uvrA Gene Transcription by LexA Protein */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants lack one or more of the Uvr proteins and are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the presumed positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, where the DNA was either irradiated or non-irradiated. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and non-irradiated DNA, while UvrB would bind to DNA only when the following conditions are present at once: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4 C and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4 C. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

==Regulation==
===Regulation of uvrA Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-07T19:05:21Z

Mariewong: /* Regulation */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants lack one or more of the Uvr proteins and are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the presumed positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, where the DNA was either irradiated or non-irradiated. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and non-irradiated DNA, while UvrB would bind to DNA only when the following conditions are present at once: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4 C and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4 C. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

==Regulation==
===Regulation Through Inhibition of uvrA Gene Transcription by LexA Protein===
Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-07T19:01:53Z

Mariewong: /* Regulation */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides using (A)BC exinuclease. This complex is made up of proteins UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants lack one or more of the Uvr proteins and are very sensitive to killing by UV irradiation because they are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is responsible for first scanning DNA for distortions, such as those caused by a thymine dimer or another chemical adduct on a base. Once a genome distortion is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to form a bubble.<ref name='PMID:7713940'/> UvrA must dissociate from the UvrB-DNA complex before UvrC can bind to the complex. Once UvrC joins the complex, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to replace the excised DNA, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations were made in the helix-turn-helix regions of two mutant UvrA proteins, G502D, and V508D by degenerate oligonucleotide-directed mutagenesis. These proteins carry mutations at the presumed positions of the helix-turn-helix motif, and so were chosen for purification and classification. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, where the DNA was either irradiated or non-irradiated. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and protein content was determined by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and non-irradiated DNA, while UvrB would bind to DNA only when the following conditions are present at once: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4 C and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4 C. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

==Regulation==

Plasmid pDR1996 carries both uvrA and ssb genes, and so was used to in an experiment to study uvrA regulation. Location of ssb was determined from DNA and protein sequencing, and subcloning and transcription experiments, such as the removal of a fragment inactivating uvrA, thus determining the site as that of uvrA, were used to locate the uvrA promoter. This fragment was also useful in establishing the nucleotide sequence of the uvrA promoter.

LexA was included in the transcription reactions and was found to inhibit transcription of the uvrA gene. By specificially binding to the promoter-operator region of uvrA , LexA guards the uvrA promoter from an overlapping segment which would result in DNase I digestion. The segment was found to be sequentially homologous with the LexA binding sites found in the recA, lexA, and uvrB genes. It was shown that uvrA’s transcription was inhibited by LexA due to its operator similar to SOS genes.<ref name='PMID:6283374'/>

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-05-07T19:00:34Z

Mariewong: /* UvrB Delivery to Damaged Sites in DNA by UvrA */

Excision Repair by the Uvr System

2012-05-07T18:52:20Z

Mariewong: /* DNA Damage Recognition by UvrA */

Excision Repair by the Uvr System

2012-05-07T18:41:50Z

Mariewong: /* General Description */

Excision Repair by the Uvr System

2012-04-19T23:02:15Z

Mariewong: /* UvrB Delivery to Damaged Sites in DNA by UvrA */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides and determines the correct sequence from the complementary DNA strand using (A)BC exinuclease. This complex is made up of a few proteins, UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants of the uvr genes are very sensitive to killing by UV irradiation and are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is the first of four proteins that largely assists in excision repair and is responsible for first scanning DNA for distortions. Once a genome distortion, such as those caused by a thymine dimer or another chemical adduct on a base, is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to a bubble.<ref name='PMID:7713940'/> It is necessary that UvrA dissociates from the UvrB-DNA complex before UvrC can bind to the complex. Once it does, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to fill the gap that results from the excision, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations by degenerate oligonucleotide-directed mutagenesis were created in these regions of two mutant UvrA proteins, G502D and V508D. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, and both types were either non-irradiated or irradiated with UV light. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and determined for protein content by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and nonirradiated DNA, while UvrB would bind to DNA only under a few conditions: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4 C and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4 C. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

==Regulation==

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-04-16T23:33:14Z

Mariewong: /* Function */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides and determines the correct sequence from the complementary DNA strand using (A)BC exinuclease. This complex is made up of a few proteins, UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants of the uvr genes are very sensitive to killing by UV irradiation and are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is the first of four proteins that largely assists in excision repair and is responsible for first scanning DNA for distortions. Once a genome distortion, such as those caused by a thymine dimer or another chemical adduct on a base, is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to a bubble.<ref name='PMID:7713940'/> It is necessary that UvrA dissociates from the UvrB-DNA complex before UvrC can bind to the complex. Once it does, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to fill the gap that results from the excision, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations by degenerate oligonucleotide-directed mutagenesis were created in these regions of two mutant UvrA proteins, G502D and V508D. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, and both types were either non-irradiated or irradiated with UV light. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and determined for protein content by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and nonirradiated DNA, while UvrB would bind to DNA only under a few conditions: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly (Table 2) when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

===DNA Incision by UvrB and UvrC===
Chromatography was performed to separate the ATP that is required in the UvrB-binding reaction and was done so after UvrB-DNA complex formation. An initial step in determining nucleotide binding and/or hydrolysis reactions in the incision of DNA upon addition of UvrC, the chromatography was carried out in the absence of ATP at 4 C and resulted in the isolation of UvrB-DNA complexes. The yield suggests that the complexes may contain unhydrolyzed ATP or ADP molecules which do not dissociate at the temperature of which the experiment was performed.

To examine whether or not ATP was hydrolyzed, the UvrB was then combined with reaction mixtures consisting of either [α-phosphate]ATP or [γ-phosphate]ATP, and the UvrB-DNA complexes were once again isolated via column chromatography using buffer without nucleotide cofactor at 4 C. The amount of nucleotide associated with the complexes was measured by densitometry of silver-stained gels while the state, ATP or ADP, was determined by scintillation counting with use of the phosphate radiolabels present. It was shown that the amount of UvrB complexed with DNA was about 5- to 10-fold higher than that complexed with nucleotide, indicating that there is no associated nucleotide cofactor associated with UvrB-DNA complexes.

Regarding the UvrB-DNA complexes, the nonessential nucleotide cofactor element allowed for the study of its dependence on the action of incision: only when in the presence of UvrC as well as ATP did the DNA incise. Additionally, in both the presence and absence of UvrC, ATP hydrolysis by the complex did not occur, as it is not required for incision. The use of ADP as a nucleotide cofactor resulted in a low level of incision and GTP showed to slightly inhibit ATP-dependent incision, so neither ADP nor GTP stimulated incision. The catalysis of DNA incision by UvrB and UvrC requires only ATP binding.<ref name='PMID:2168423'/>

==Regulation==

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

PMID:1387640

2012-04-13T20:38:54Z

Mariewong: Fill PMID: Page!

{{RightTOC}}


{| id="Q4f888ede86cd1" class=" tableEdit PMID_info_table"

|-
!align=left |Citation
||
'''Lin, JJ, Phillips, AM, Hearst, JE and Sancar, A''' (1992) Active site of (A)BC excinuclease. II. Binding, bending, and catalysis mutants of UvrB reveal a direct role in 3' and an indirect role in 5' incision.''J. Biol. Chem.'' '''267''':17693-700
|-
!align=left |Abstract
||
UvrB plays a central role in (A)BC excinuclease. To study its role in the incision reactions, conserved His and Asp residues in this subunit were mutagenized. All His and the majority of Asp mutants behaved like wild-type protein in vivo and in vitro. However, three mutants, D337A, D478A, and D510A, either completely or partially abolished UvrB activity. All three mutant proteins associate with UvrA normally but D337A and D510A were unable to bind to DNA specifically. The UvrB-D478A mutant bound to DNA specifically but failed to denature and kink the DNA. However, UvrB-D478A was efficiently loaded onto DNA preincised at the 3' site and promoted near-normal incision by UvrC at the 5' site. We propose that D478 is involved in bending DNA and catalysis of the 3' incision and that the 3' incision precedes the 5' incision. UvrB which is missing the carboxyl-terminal 43 amino acids binds to, and kinks DNA but is unable to make the 3' incision suggesting that it is missing a residue involved in catalysis. This residue was identified to be E639 by site-specific mutagenesis.
|-
!align=left |Links
||
[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=1387640 PubMed]

|-
!align=left |Keywords
||
Adenosine Triphosphatases; Bacterial Proteins; Binding Sites; DNA Helicases; DNA, Bacterial; DNA-Binding Proteins; Dose-Response Relationship, Radiation; Endodeoxyribonucleases; Escherichia coli; Escherichia coli Proteins; Genes, Bacterial; Kinetics; Macromolecular Substances; Models, Structural; Mutagenesis, Site-Directed; Nucleic Acid Conformation; Ultraviolet Rays

|- class="tableEdit_footer"
|[{{SERVER}}{{SCRIPTPATH}}?title=Special:TableEdit&id=d41d8cd98f00b204e9800998ecf8427e.181259.Q4f888ede86cd1&page=181259&pagename={{FULLPAGENAMEE}}&type=1&template=PMID_info_table edit table] ||
|}


==Significance {{HelpIcon|Help:Literature}} ==
{{LitSignificance}}

== Useful Materials and Methods {{HelpIcon|Help:Literature}}==
{{LitMaterials}}

==Annotations {{HelpIcon|Help:Literature}}==
<protect>

{| id="F4f888ede8cd86" class=" tableEdit GO_table_reference dataTable"
|- {{table heading style}}
!|Gene product!!Qualifier!!GO ID!!GO term name!!Evidence Code!!with/from!!Aspect!!Notes!!Status

|- class="tableEdit_footer"
|[{{SERVER}}{{SCRIPTPATH}}?title=Special:TableEdit&id=d41d8cd98f00b204e9800998ecf8427e.181259.F4f888ede8cd86&page=181259&pagename={{FULLPAGENAMEE}}&type=0&template=GO_table_reference edit table] || || || || || || || ||
|}
</protect>

==EcoliWiki Links {{HelpIcon|Help:Literature}} ==
{{LitLinks}}

==See also==

==References==
{{RefHelp}}
<references/>

PMID:1387640

2012-04-13T20:38:54Z

Mariewong: New PMID: Page!

PMID:7713940

2012-04-13T20:38:10Z

Mariewong: Fill PMID: Page!

{{RightTOC}}


{| id="W4f888eb235d32" class=" tableEdit PMID_info_table"

|-
!align=left |Citation
||
'''Hsu, DS, Kim, ST, Sun, Q and Sancar, A''' (1995) Structure and function of the UvrB protein.''J. Biol. Chem.'' '''270''':8319-27
|-
!align=left |Abstract
||
UvrB plays a central role in (A)BC excinuclease. To identify the regions of UvrB which are involved in interacting with UvrA, UvrC, and DNA, deletion mutants, point mutants, and various fusion forms of UvrB were constructed and characterized. We found that the region encompassing amino acid residues 115-250 of UvrB binds to UvrA, while the region encompassing amino acid residues 547-673 binds to both UvrA and UvrC. In addition, the region between these two domains, which contains the helicase motifs II-VI, was found to be involved in binding to DNA. Within this DNA-binding region, two point mutants, E265A and E338A, were found to be unable to bind DNA while two residues, Phe-365 and Phe-496, were identified to interact with DNA. Furthermore, fluorescence quenching studies with mutants F365W and F496W and repair of thymine cyclobutane dimers by photoinduced electron transfer by these mutants suggest that residues Phe-365 and Phe-496 interact with DNA most likely through stacking interactions.
|-
!align=left |Links
||
[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=7713940 PubMed]

|-
!align=left |Keywords
||
Adenosine Triphosphatases; Bacterial Proteins; Base Sequence; Cross-Linking Reagents; DNA; DNA Helicases; DNA-Binding Proteins; Endodeoxyribonucleases; Escherichia coli Proteins; Ficusin; Hydrolysis; Molecular Sequence Data; Oligodeoxyribonucleotides; Point Mutation; Protein Binding; Pyrimidine Dimers; Sequence Deletion; Spectrometry, Fluorescence; Structure-Activity Relationship

|- class="tableEdit_footer"
|[{{SERVER}}{{SCRIPTPATH}}?title=Special:TableEdit&id=d41d8cd98f00b204e9800998ecf8427e.181258.W4f888eb235d32&page=181258&pagename={{FULLPAGENAMEE}}&type=1&template=PMID_info_table edit table] ||
|}


==Significance {{HelpIcon|Help:Literature}} ==
{{LitSignificance}}

== Useful Materials and Methods {{HelpIcon|Help:Literature}}==
{{LitMaterials}}

==Annotations {{HelpIcon|Help:Literature}}==
<protect>

{| id="G4f888eb24b98b" class=" tableEdit GO_table_reference dataTable"
|- {{table heading style}}
!|Gene product!!Qualifier!!GO ID!!GO term name!!Evidence Code!!with/from!!Aspect!!Notes!!Status

|- class="tableEdit_footer"
|[{{SERVER}}{{SCRIPTPATH}}?title=Special:TableEdit&id=d41d8cd98f00b204e9800998ecf8427e.181258.G4f888eb24b98b&page=181258&pagename={{FULLPAGENAMEE}}&type=0&template=GO_table_reference edit table] || || || || || || || ||
|}
</protect>

==EcoliWiki Links {{HelpIcon|Help:Literature}} ==
{{LitLinks}}

==See also==

==References==
{{RefHelp}}
<references/>

PMID:7713940

2012-04-13T20:38:10Z

Mariewong: New PMID: Page!

Excision Repair by the Uvr System

2012-04-13T20:34:51Z

Mariewong: /* UvrB Delivery to Damaged Sites in DNA by UvrA */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides and determines the correct sequence from the complementary DNA strand using (A)BC exinuclease. This complex is made up of a few proteins, UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants of the uvr genes are very sensitive to killing by UV irradiation and are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is the first of four proteins that largely assists in excision repair and is responsible for first scanning DNA for distortions. Once a genome distortion, such as those caused by a thymine dimer or another chemical adduct on a base, is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to a bubble.<ref name='PMID:7713940'/> It is necessary that UvrA dissociates from the UvrB-DNA complex before UvrC can bind to the complex. Once it does, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to fill the gap that results from the excision, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations by degenerate oligonucleotide-directed mutagenesis were created in these regions of two mutant UvrA proteins, G502D and V508D. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, and both types were either non-irradiated or irradiated with UV light. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
The delivery of UvrB to damaged DNA by UvrA was examined by first incubating UvrA and UvrB with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and determined for protein content by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and nonirradiated DNA, while UvrB would bind to DNA only under a few conditions: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly (Table 2) when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

==Regulation==

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-04-12T00:05:12Z

Mariewong: /* Function */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides and determines the correct sequence from the complementary DNA strand using (A)BC exinuclease. This complex is made up of a few proteins, UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants of the uvr genes are very sensitive to killing by UV irradiation and are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is the first of four proteins that largely assists in excision repair and is responsible for first scanning DNA for distortions. Once a genome distortion, such as those caused by a thymine dimer or another chemical adduct on a base, is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to a bubble.<ref name='PMID:7713940'/> It is necessary that UvrA dissociates from the UvrB-DNA complex before UvrC can bind to the complex. Once it does, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to fill the gap that results from the excision, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations by degenerate oligonucleotide-directed mutagenesis were created in these regions of two mutant UvrA proteins, G502D and V508D. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, and both types were either non-irradiated or irradiated with UV light. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
To study the delivery of UvrB to damaged DNA by UvrA, UvrA and UvrB were first incubated with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and determined for protein content by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and nonirradiated DNA, while UvrB would bind to DNA only under a few conditions: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly (Table 2) when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

==Regulation==

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-04-12T00:04:45Z

Mariewong: /* UvrB Delivery to Damaged Sites in DNA by UvrA */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides and determines the correct sequence from the complementary DNA strand using (A)BC exinuclease. This complex is made up of a few proteins, UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants of the uvr genes are very sensitive to killing by UV irradiation and are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is the first of four proteins that largely assists in excision repair and is responsible for first scanning DNA for distortions. Once a genome distortion, such as those caused by a thymine dimer or another chemical adduct on a base, is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to a bubble.<ref name='PMID:7713940'/> It is necessary that UvrA dissociates from the UvrB-DNA complex before UvrC can bind to the complex. Once it does, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to fill the gap that results from the excision, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations by degenerate oligonucleotide-directed mutagenesis were created in these regions of two mutant UvrA proteins, G502D and V508D. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, and both types were either non-irradiated or irradiated with UV light. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
To study the delivery of UvrB to damaged DNA by UvrA, UvrA and UvrB were first incubated with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and determined for protein content by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and nonirradiated DNA, while UvrB would bind to DNA only under a few conditions: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly (Table 2) when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.<ref name='PMID:2546148'/>

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

==Regulation==

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-04-12T00:02:19Z

Mariewong: /* Function */

==General Description==
Excision repair by the Uvr System is a DNA repair mechanism that removes a wide variety of damaged nucleotides and determines the correct sequence from the complementary DNA strand using (A)BC exinuclease. This complex is made up of a few proteins, UvrA, UvrB, and UvrC, which carry out coordinated actions to complete the excision repair. Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants of the uvr genes are very sensitive to killing by UV irradiation and are incapable of removing thymine-thymine and thymine-cytosine adducts.

UvrA is the first of four proteins that largely assists in excision repair and is responsible for first scanning DNA for distortions. Once a genome distortion, such as those caused by a thymine dimer or another chemical adduct on a base, is detected by UvrA, UvrA guides UvrB to the lesion in the DNA. The DNA is bent by approximately 130° and UvrB proceeds to melt the DNA, unwinding the area around the lesion by about 5bp to a bubble.<ref name='PMID:7713940'/> It is necessary that UvrA dissociates from the UvrB-DNA complex before UvrC can bind to the complex. Once it does, the 3’ incision is made by UvrB while the 5’ incision is made by UvrC.<ref name='PMID:1387640'/> This cleavage excises the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. DNA polymerase uses the undamaged, complementary strand as a template to fill the gap that results from the excision, and DNA ligase forms covalent phosphodiester bonds between the 3’ hydroxyl ends of one nucleotide with the 5’ phosphate end of another. Once complete, the original nucleotide sequence is restored.

==Components==

==Function==
===DNA Damage Recognition by UvrA===
To study the helix-turn-helix motifs of UvrA proteins, random mutations by degenerate oligonucleotide-directed mutagenesis were created in these regions of two mutant UvrA proteins, G502D and V508D. The DNA binding of wild-type UvrA proteins was then compared with that of the mutant UvrA proteins, and both types were either non-irradiated or irradiated with UV light. It was shown that mutations in the helix-turn-helix motif region of a UvrA protein decreases the protein’s binding affinity for DNA by decreasing its ability to recognize DNA damage.<ref name='PMID:8444906'/>

===UvrB Delivery to Damaged Sites in DNA by UvrA===
To study the delivery of UvrB to damaged DNA by UvrA, UvrA and UvrB were first incubated with UV-irradiated or nonirradiated DNA in the presence of ATP. The resulting DNA-protein complexes were isolated by size-exclusion chromatography, and determined for protein content by SDS/PAGE with silver staining and quantitative densitometry afterwards. It was found that UvrA would bind to both UV-irradiated and nonirradiated DNA, while UvrB would bind to DNA only under a few conditions: when DNA was UV-irradiated, when UvrA was present in the reaction mixture, and when ATP was present in the reaction mixture, as well as in the column buffer. This reflects the affinity of UvrA for DNA in addition to the formation specifics of the UvrA-UvrB complex in which UvrA is dimerized and interacts with UvrB through an ATP-stimulated reaction. The ratio of UvrA to UvrB bound to DNA drastically changed, approximately by 5-fold more, when the ratio of UvrA to UvrB in the reaction mixture varied, once again showing the strong affinity of UvrA for DNA. On the other hand, the ratio of the DNA-protein complex decreased significantly (Table 2) when the UvrA to UvrB ratio in the reaction mixture decreased, indicating that the binding of UvrB to UV-irradiated DNA is promoted by UvrA catalytically instead of stoichiometrically.

===UvrB and UvrC Interaction===
Affinity columns were used to study the possible direct interaction between UvrB and UvrC because they are able to detect weak, but specific protein-protein interactions. First, ''E. coli'' cell-free extract from a strain overproducing the UvrB protein was passed through UvrC affinity columns. UvrB protein remained, and so did RNA polymerase. The specificity of the results was then inspected by use of a control column without any cross-linked protein. Here, while UvrB did not bind to the column, some RNA polymerase continued to do so, suggesting that the binding of UvrB to UvrC is specific, while that of RNA polymerase to UvrC is nonspecific. To determine that the UvrB-UvrC interaction seen here was specific, E.coli cell-free extract from a strain overproducing UvrC protein was passed through a UvrB affinity column instead. Only UvrC was retained on the column. Although with lower affinity than the UvrA-UvrB interaction, it was concluded that UvrB and UvrC interact off DNA specifically.<ref name='PMID:7713940'/>

==Regulation==

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-03-27T23:53:54Z

Mariewong: /* DNA Damage Recognition by UvrA */

Excision Repair by the Uvr System

2012-03-27T23:51:44Z

Mariewong: /* DNA Damage Recognition by UvrA */

Excision Repair by the Uvr System

2012-03-27T23:50:31Z

Mariewong: /* DNA Damage Recognition by UvrA */

Excision Repair by the Uvr System

2012-03-27T23:49:08Z

Mariewong: /* UvrB and UvrC Interaction */

Excision Repair by the Uvr System

2012-03-08T23:59:45Z

Mariewong: /* Function */

Excision Repair by the Uvr System

2012-03-08T23:54:32Z

Mariewong: /* General Description */

Excision Repair by the Uvr System

2012-03-08T23:52:26Z

Mariewong: /* General Description */

Excision Repair by the Uvr System

2012-03-08T23:50:36Z

Mariewong: /* General Description */

Excision Repair by the Uvr System

2012-02-24T23:06:05Z

Mariewong:

==General Description==
UvrA and UvrB are the first of four proteins that largely assist in excision repair, and are both responsible for first scanning DNA for distortions. Once a genome distortion, such as those caused by a thymine dimer or another chemical adduct on a base, is detected by UvrA, UvrA leaves and UvrB proceeds to melt the DNA, creating a bubble around the lesion. UvrB then recruits UvrC. UvrC cleaves the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. The gap which results from the excision is filled by DNA polymerase and DNA ligase using the undamaged strand as a template. Once complete, the original nucleotide sequence is restored.

Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants of the ''uvr'' genes are very sensitive to killing by UV irradiation and are incapable of removing thymine-thymine and thymine-cystine adducts.
==Components==

==Function==

==Regulation==

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-02-24T23:02:40Z

Mariewong:

==General Description==
UvrA and UvrB are the first of four enzymes that assist in excision repair, and are both responsible for first scanning DNA for distortions. Once a genome distortion, such as those caused by a thymine dimer or another chemical adduct on a base, is detected by UvrA, UvrA leaves and UvrB proceeds to melt the DNA, creating a bubble around the lesion. UvrB then recruits UvrC. UvrC cleaves the area of the bubble, including the lesion, creating a short, single-stranded DNA segment that is then removed by DNA helicase, UvrD. The gap which results from the excision is filled by DNA polymerase and DNA ligase using the undamaged strand as a template. Once complete, the original nucleotide sequence is restored.

Uvr proteins are necessary to repair damage caused by ultraviolet light in genomes. Mutants of the ''uvr'' genes are very sensitive to killing by UV irradiation and are incapable of removing thymine-thymine and thymine-cystine adducts.
==Components==

==Function==

==Regulation==

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-02-24T22:58:04Z

Mariewong: Undo revision 1369565 by Mariewong (talk)

==General Description==

==Components==

==Function==

==Regulation==

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]

Excision Repair by the Uvr System

2012-02-24T22:57:46Z

Mariewong: Undo revision 1369564 by Mariewong (talk)

==General Description=

==Components==

==Function==

==Regulation==

==Cell Biology==

==Experimental Resources==

==Comparison with other Organisms==

==Notes==
{{UserContentHelp}}

==Related GO Terms ==
[[Help:Gene Ontology]]

==References==
{{RefHelp}}
<references/>

__NOTOC__
[[Category:Processes]]