Excision Repair by the Uvr System

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. 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. 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.

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.

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.

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.

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.

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.

Notes

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Related GO Terms

Help:Gene Ontology

References

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