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Excision Repair by the Uvr System

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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.[1] 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.[2] 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.



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.[3]

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.[4]

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.[1]

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.[5]

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.[6]

Tomko et al.[7] 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.[7]


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.[8]

Cell Biology

Experimental Resources

Comparison with other Organisms


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  1. 1.0 1.1 Hsu, DS et al. (1995) Structure and function of the UvrB protein. J. Biol. Chem. 270 8319-27 PubMed EcoliWiki page
  2. Lin, JJ et al. (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 PubMed EcoliWiki page
  3. Wang, J & Grossman, L (1993) Mutations in the helix-turn-helix motif of the Escherichia coli UvrA protein eliminate its specificity for UV-damaged DNA. J. Biol. Chem. 268 5323-31 PubMed EcoliWiki page
  4. Orren, DK & Sancar, A (1989) The (A)BC excinuclease of Escherichia coli has only the UvrB and UvrC subunits in the incision complex. Proc. Natl. Acad. Sci. U.S.A. 86 5237-41 PubMed EcoliWiki page
  5. Orren, DK & Sancar, A (1990) Formation and enzymatic properties of the UvrB.DNA complex. J. Biol. Chem. 265 15796-803 PubMed EcoliWiki page
  6. Maluf, NK & Lohman, TM (2003) Self-association equilibria of Escherichia coli UvrD helicase studied by analytical ultracentrifugation. J. Mol. Biol. 325 889-912 PubMed EcoliWiki page
  7. 7.0 7.1 Tomko, EJ et al. (2012) Single-stranded DNA translocation of E. coli UvrD monomer is tightly coupled to ATP hydrolysis. J. Mol. Biol. 418 32-46 PubMed EcoliWiki page
  8. Sancar, A et al. (1982) LexA protein inhibits transcription of the E. coli uvrA gene in vitro. Nature 298 96-8 PubMed EcoliWiki page