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 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. 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. 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.

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.

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.

Notes

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Related GO Terms

Help:Gene Ontology

References

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