Single-stranded DNA-binding Protein Recruits DNA Polymerase V to Primer Termini on RecA-coated DNA

Abstract

Translesion DNA synthesis (TLS) by DNA polymerase V (polV) in Escherichia coli involves accessory proteins, including RecA and single-stranded DNA-binding protein (SSB). To elucidate the role of SSB in TLS we used an in vitro exonuclease protection assay and found that SSB increases the accessibility of 3′ primer termini located at abasic sites in RecA-coated gapped DNA. The mutant SSB-113 protein, which is defective in protein-protein interactions, but not in DNA binding, was as effective as wild-type SSB in increasing primer termini accessibility, but deficient in supporting polV-catalyzed TLS. Consistently, the heterologous SSB proteins gp32, encoded by phage T4, and ICP8, encoded by herpes simplex virus 1, could replace E. coli SSB in the TLS reaction, albeit with lower efficiency. Immunoprecipitation experiments indicated that polV directly interacts with SSB and that this interaction is disrupted by the SSB-113 mutation. Taken together our results suggest that SSB functions to recruit polV to primer termini on RecA-coated DNA, operating by two mechanisms: 1) increasing the accessibility of 3′ primer termini caused by binding of SSB to DNA and 2) a direct SSB-polV interaction mediated by the C terminus of SSB. Translesion DNA synthesis (TLS) by DNA polymerase V (polV) in Escherichia coli involves accessory proteins, including RecA and single-stranded DNA-binding protein (SSB). To elucidate the role of SSB in TLS we used an in vitro exonuclease protection assay and found that SSB increases the accessibility of 3′ primer termini located at abasic sites in RecA-coated gapped DNA. The mutant SSB-113 protein, which is defective in protein-protein interactions, but not in DNA binding, was as effective as wild-type SSB in increasing primer termini accessibility, but deficient in supporting polV-catalyzed TLS. Consistently, the heterologous SSB proteins gp32, encoded by phage T4, and ICP8, encoded by herpes simplex virus 1, could replace E. coli SSB in the TLS reaction, albeit with lower efficiency. Immunoprecipitation experiments indicated that polV directly interacts with SSB and that this interaction is disrupted by the SSB-113 mutation. Taken together our results suggest that SSB functions to recruit polV to primer termini on RecA-coated DNA, operating by two mechanisms: 1) increasing the accessibility of 3′ primer termini caused by binding of SSB to DNA and 2) a direct SSB-polV interaction mediated by the C terminus of SSB. Translesion DNA synthesis is a DNA damage tolerance mechanism in which replication blocks caused by DNA lesions are relieved by specialized DNA polymerases proficient in synthesizing DNA across lesions (1Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. 2nd Ed. DNA Repair and Mutagenesis. ASM Press, Washington DC2006: 463-661Google Scholar). In Escherichia coli this reaction is catalyzed primarily by the Y family DNA polymerases encoded by the umuDC operon (DNA polymerase V (polV) 2The abbreviations used are:polVpolymerase VpolIVpolymerase IVSSBsingle-stranded DNA-binding proteinHSVherpes simplex virusMBPmaltose-binding proteinssDNAsingle-stranded DNATLStranslesion DNA synthesisRPAreplication protein A. 2The abbreviations used are:polVpolymerase VpolIVpolymerase IVSSBsingle-stranded DNA-binding proteinHSVherpes simplex virusMBPmaltose-binding proteinssDNAsingle-stranded DNATLStranslesion DNA synthesisRPAreplication protein A.) and the dinB gene (DNA polymerase IV (polIV)) (2Goodman M.F. Trends Biochem. Sci. 2000; 25: 189-195Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 3Livneh Z. J. Biol. Chem. 2001; 276: 25639-25642Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 4Wagner J. Etienne H. Janel-Bintz R. Fuchs R.P. DNA Repair. 2002; 2: 159-167Crossref Scopus (78) Google Scholar, 5Jarosz D.F. Beuning P.J. Cohen S.E. Walker G.C. Trends Microbiol. 2007; 15: 70-77Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The in vitro reconstitution of TLS with purified proteins indicated that both RecA and single-stranded DNA-binding protein (SSB) are required for polV-catalyzed TLS (6Tang M. Bruck I. Eritja R. Turner J. Frank E.G. Woodgate R. O'Donnell M. Goodman M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9755-9760Crossref PubMed Scopus (180) Google Scholar, 7Reuven N.B. Tomer G. Livneh Z. Mol. Cell. 1998; 2: 191-199Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 8Reuven N.B. Arad G. Maor-Shoshani A. Livneh Z. J. Biol. Chem. 1999; 274: 31763-31766Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 9Tang M. Shen X. Frank E.G. O'Donnell M. Woodgate R. Goodman M.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8919-8924Crossref PubMed Scopus (487) Google Scholar). RecA, the main recombinase in E. coli, is also the main activator of the SOS stress response via its ability to promote the autocleavage of the LexA repressor, which is the negative regulator of all SOS genes. RecA has two additional roles in TLS: (a) It activates UmuD by promoting its autocleavage to UmuD′, which then binds to UmuC to form the UmuD′2C complex polV (1Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. 2nd Ed. DNA Repair and Mutagenesis. ASM Press, Washington DC2006: 463-661Google Scholar). (b) A direct role in TLS which involves the recruitment of UmuD′ to DNA (10Frank E.G. Hauser J. Levine A.S. Woodgate R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8169-8173Crossref PubMed Scopus (97) Google Scholar). RecA promotes these activities in its DNA-bound form, which consists of a nucleoprotein filament formed by its cooperative binding to ssDNA (11Roca A.I. Cox M.M. CRC Crit. Rev. Biochem. Mol. Biol. 1990; 25: 415-456Crossref PubMed Scopus (363) Google Scholar). polymerase V polymerase IV single-stranded DNA-binding protein herpes simplex virus maltose-binding protein single-stranded DNA translesion DNA synthesis replication protein A. polymerase V polymerase IV single-stranded DNA-binding protein herpes simplex virus maltose-binding protein single-stranded DNA translesion DNA synthesis replication protein A. SSB is an essential protein in E. coli, involved in replication, recombination and repair. Functional homologs of SSB are present in all organisms, and their importance is well illustrated by the occurrence of viral SSBs (12Lohman T.M. Bujalowski W. Overman L.B. Trends Biochem. Sci. 1988; 13: 250-255PubMed Google Scholar, 13Meyer R.R. Laine P.S. Microbiol. Rev. 1990; 54: 342-380Crossref PubMed Google Scholar, 14Kornberg A. Baker T. DNA Replication. Second Ed., W. H. and Company, New York1991Google Scholar, 15Lohman T.M. Ferrari M.E. Annu. Rev. Biochem. 1994; 63: 527-570Crossref PubMed Scopus (528) Google Scholar). E. coli SSB is a 75kDa tetramer composed of four identical subunits. It binds ssDNA strongly via a ssDNA-binding domain located at its N terminus, and interacts with a variety of proteins via its C terminus (15Lohman T.M. Ferrari M.E. Annu. Rev. Biochem. 1994; 63: 527-570Crossref PubMed Scopus (528) Google Scholar, 16Genschel J. Curth U. Urbanke C. Biol. Chem. 2000; 381: 183-192Crossref PubMed Scopus (99) Google Scholar, 17Sandigursky M. Mendez F. Bases R.E. Matsumoto T. Franklin W.A. Radiat. Res. 1996; 145: 619-623Crossref PubMed Scopus (33) Google Scholar, 18Cadman C.J. McGlynn P. Nucleic Acids Res. 2004; 32: 6378-6387Crossref PubMed Scopus (131) Google Scholar, 19Kelman Z. Yuzhakov A. Andjelkovic J. O'Donnell M. EMBO J. 1998; 17: 2436-2449Crossref PubMed Scopus (155) Google Scholar, 20Witte G. Urbanke C. Curth U. Nucleic Acids Res. 2004; 31: 4434-4440Crossref Scopus (88) Google Scholar). A detailed kinetic analysis showed that SSB had a dramatic effect on TLS by polV across an abasic site (21Pham P. Bertram J.G. O'Donnell M. Woodgate R. Goodman M.F. Nature. 2001; 409: 366-370Crossref PubMed Scopus (110) Google Scholar), however, under some conditions SSB was found to be stimulatory rather than essential for in vitro TLS (22Fujii S. Gasser V. Fuchs R.P. J. Mol. Biol. 2004; 341: 405-417Crossref PubMed Scopus (60) Google Scholar). We used an in vitro reconstituted TLS assay system to study the mechanism by which SSB exerts its effect on TLS. We report here that SSB has a dual role in polV-catalyzed TLS: 1) it increases the accessibility of the primer-template near a template lesion on RecA-coated DNA, and 2) it directly interacts with UmuC. This suggests that SSB functions in the recruitment of polV to the primer-template-lesion site on RecA-coated DNA, an early stage in the initiation of TLS. Proteins—Proteins used in this study were purified as described in the respective references: RecA (23Cox M.M. McEntee K. Lehman I.R. J. Biol. Chem. 1981; 256: 4676-4678Abstract Full Text PDF PubMed Google Scholar); E. coli SSB mutant proteins H55Y (SSB-1), H55K, and P176S (SSB-113) U. I. J. F. Urbanke C. G. J. Biochem. PubMed Scopus Google Scholar); polV and UmuD′ purified N.B. Tomer G. Livneh Z. Mol. Cell. 1998; 2: 191-199Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 8Reuven N.B. Arad G. Maor-Shoshani A. Livneh Z. J. Biol. Chem. 1999; 274: 31763-31766Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar); E. coli SSB T.M. Overman L.B. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T.M. 25: PubMed Scopus Google Scholar), with an additional of a the of J. Biol. Chem. Full Text PDF PubMed Google Scholar). simplex virus was a I. Lehman of and the complex and were O'Donnell New gene protein, E. coli DNA and E. coli exonuclease were and were DNA were New exonuclease was and was and were of E. coli as by of gapped were as described G. N.B. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar, G. Livneh Z. 1999; PubMed Scopus Google Scholar). a primer was together with to the and of this gapped to the the reaction were by The gapped was the an and The was to a of phage exonuclease as described G. N.B. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). TLS TLS reaction was as described N.B. Tomer G. Livneh Z. Mol. Cell. 1998; 2: 191-199Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, G. N.B. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar), with The reaction and of and gapped SSB RecA, polV and the was at and the complex was at SSB H55K, SSB-113 were at was at and at were at as RecA, and the complex and were with the DNA in the assay for the SSB was and were for additional polV was and the reaction was for were and the DNA were as described N.B. Arad G. A. Livneh Z. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The of was by the of by the of the TLS was also by the of by the of DNA. of synthesis was by the of by the of DNA. primer accessibility assay of the of the 3′ by E. coli exonuclease as a RecA-coated were under conditions to the TLS but and and in the of E. coli exonuclease at proteins, were at the RecA, E. coli wild-type SSB and gp32, the RecA protein was present at a the assay was in the of an of and were at as RecA and the DNA were in the assay for were and the were for exonuclease was and the reaction was at for were with and by as described for the TLS protection assay the of the of the by RecA and SSB. The sites are in The were as described for the TLS assay with the The DNA used were not and were with and with RecA E. coli SSB as and an as described a in to the was This is a which is to that it is and lesion S. S. G. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). were at as RecA and the DNA at were in the assay for SSB was at this The were for which the was and the reaction was at for to were by the of for at and by in the of were under Immunoprecipitation were by the indicated proteins and at for in a and in a of A New in of binding was then to reaction for an additional of at protein A was to the reaction for an additional of The protein A were with of binding proteins were by in of a and and by The of SSB in the was by with G. Urbanke C. Curth U. Nucleic Acids Res. PubMed Scopus Google Scholar), by to was used for on The and SSB in polV-catalyzed in vitro TLS assay used in this which was in our N.B. Tomer G. Livneh Z. Mol. Cell. 1998; 2: 191-199Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 8Reuven N.B. Arad G. Maor-Shoshani A. Livneh Z. J. Biol. Chem. 1999; 274: 31763-31766Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, G. Livneh Z. 1999; PubMed Scopus Google Scholar), is on a gapped a abasic site in the ssDNA and an near the primer of RecA, and DNA synthesis the 3′ primer terminus, and to and across the abasic To the analysis of TLS the DNA is the reaction and to a of replication at the and TLS of the in this the of as (a) as all of all (b) as all synthesis than of all that were lesion is at with to the and as all than of all we the mutant in vitro polV-catalyzed TLS. The mutant a in as well as J. R.R. A. J. PubMed Google Scholar, Mol. 1981; PubMed Scopus Google Scholar). In vitro analysis that the a in of the protein, which is to be for its J. Biol. Chem. Full Text PDF PubMed Google Scholar). of TLS with RecA, and the protein in both synthesis and by polV Consistently, analysis of the SSB mutant protein, to be defective than in in vitro U. I. J. F. Urbanke C. G. J. Biochem. PubMed Scopus Google Scholar), showed lower of initiation and by polV results suggest that SSB is required in its form polV-catalyzed TLS. SSB the of the 3′ in RecA-coated RecA nucleoprotein filament was to polV to the DNA, its interaction with as the RecA filament to the DNA at the 3′ primer terminus, it with the binding of polV to the primer of RecA were found to TLS in our system A. Livneh Z. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). To directly the of RecA and SSB on primer accessibility, we an exonuclease protection on the ability of the E. coli exonuclease to the primer terminus in the be in exonuclease the primer as indicated by the of than of a RecA nucleoprotein filament with increasing of RecA caused of the of the primer terminus by exonuclease and the of the as a of exonuclease in the of RecA to in its and The of SSB relieved this to a to in primer at RecA SSB had a effect on the by exonuclease A SSB the of the in RecA-coated SSB exerts its effect via its ability to we the ability of a heterologous the phage gp32, to the accessibility of the primer terminus in RecA-coated DNA. To that we the exonuclease protection assay with RecA-coated gapped as but with of SSB. be in the of RecA to caused a in primer by exonuclease as to and of relieved this to primer had a effect on the of exonuclease and not exonuclease and The ability of to the RecA of exonuclease is to that of E. coli SSB A and that their effect is at the DNA mediated primarily by their ssDNA binding The the two SSBs their ssDNA binding We also the effect of the on primer accessibility in the of RecA, the In to the E. coli SSB and phage gp32, was to the RecA of exonuclease not SSB RecA of 3′ primer SSB of the RecA SSB a of the RecA rather a at the primer-template-lesion we used a in the has two located and to the primer of with an which and the two DNA of and of RecA at of had effect on as This that RecA, not to DNA, not the assay was with the the In the of RecA, the gapped was by two DNA the of the is than the it is the of RecA, of by was that at and RecA the of of and was also and that both sites were for the and is the single-stranded DNA these results that a nucleoprotein filament formed at the gapped RecA to the DNA to the in the both of increasing of SSB had effect on the of by as in results suggest that SSB not an of the RecA nucleoprotein It is that SSB not the of The site to the 3′ primer terminus is located To study the effect of SSB on the RecA filament to the 3′ primer terminus, was in has sites on located and to the primer terminus The of are in as Full of in the of is in the is In the of RecA, in to the DNA which was were including the of to A was the of SSB to and SSB not by of the by was by the of RecA and RecA and SSB and SSB caused an with the binding of SSB to DNA Chem. 1994; PubMed Scopus Google Scholar). The that SSB is to the RecA at sites the primer terminus and that SSB in a by the primer terminus to SSBs TLS by the of SSB in TLS involves increasing the accessibility of the 3′ primer terminus, heterologous which a be to for E. coli SSB in the in vitro TLS the results of a TLS with RecA, and in the of polV and RecA not DNA synthesis of gp32, primer by polV was to the abasic but not it and not primer the and the complex were to the TLS of polV (22Fujii S. Gasser V. Fuchs R.P. J. Mol. Biol. 2004; 341: 405-417Crossref PubMed Scopus (60) Google Scholar, M. P. Shen X. O'Donnell M. Woodgate R. Goodman M.F. Nature. 2000; PubMed Scopus Google Scholar, A. Livneh Z. 2002; PubMed Scopus Google Scholar), we their effect on TLS in the of the of these proteins in the of gp32, was to the abasic as the of the primer to a of and polV-catalyzed TLS in the of it is effective than E. coli SSB to and to A was with ICP8, the single-stranded DNA-binding protein, results DNA synthesis to the abasic but not was was present in the reaction of SSB and the and the complex were across the abasic site by polV was in the of and to the reaction in the of protein, TLS was effective E. coli SSB was by We also the effect of on polV-catalyzed TLS. In to the experiments with and ICP8, TLS was by polV in the of with its to accessibility of the 3′ primer terminus SSB in TLS via a Second to ssDNA that heterologous SSBs to a than E. coli SSB the that the by a mechanism additional to ssDNA To the that this mechanism involves protein-protein interactions, we the SSB-113 mutant protein, which was to be defective in with proteins J. Curth U. Urbanke C. Biol. Chem. 2000; 381: 183-192Crossref PubMed Scopus (99) Google Scholar, 19Kelman Z. Yuzhakov A. Andjelkovic J. O'Donnell M. EMBO J. 1998; 17: 2436-2449Crossref PubMed Scopus (155) Google Scholar), ssDNA binding J. Biol. Chem. Full Text PDF PubMed Google Scholar). A assay that our SSB-113 protein ssDNA binding The of SSB-113 on both TLS and primer accessibility were the results of a TLS in which SSB was with the SSB-113 TLS by polV was the SSB-113 wild-type SSB to we the ability of SSB-113 to the by RecA on the of exonuclease the SSB-113 protein was as effective as wild-type SSB in the by RecA to the SSB-113 protein had wild-type in increasing accessibility of the it was deficient in supporting TLS by SSB with was in our that the polV interacts with SSB Livneh Z. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). on the the two we that SSB also interacts with To this we an the of to was with which a was The was then on protein and proteins were The proteins were by and the was with be in SSB was found to the it was with binding was of the two proteins was and the was with SSB the mutant SSB-113 protein was with binding was that this the binding of SSB to The with SSB-113 showed binding, as of SSB and SSB-113 proteins were as by the E. coli polV accessory proteins for its (1Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. 2nd Ed. DNA Repair and Mutagenesis. ASM Press, Washington DC2006: 463-661Google Scholar, 3Livneh Z. J. Biol. Chem. 2001; 276: 25639-25642Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, S. R.E. Annu. Rev. Biochem. PubMed Scopus Google Scholar). A accessory protein is the RecA protein, which was to be required for in vitro in to its functions in the SOS response (1Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. 2nd Ed. DNA Repair and Mutagenesis. ASM Press, Washington DC2006: 463-661Google Scholar). RecA a nucleoprotein filament on DNA and was to recruit polV to DNA via its interaction with the UmuD′ of polV (10Frank E.G. Hauser J. Levine A.S. Woodgate R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8169-8173Crossref PubMed Scopus (97) Google Scholar). of RecA were found to polV-catalyzed TLS in our in vitro system A. Livneh Z. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), by the primer-template-lesion and the of polV to the primer terminus N.B. Arad G. A. Livneh Z. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). This not to be a of in vitro assay RecA is present at a of under SOS conditions C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The suggest that SSB this and functions to polV to the primer SSB was to be required for TLS across an abasic site by polV N.B. Arad G. Maor-Shoshani A. Livneh Z. J. Biol. Chem. 1999; 274: 31763-31766Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 9Tang M. Shen X. Frank E.G. O'Donnell M. Woodgate R. Goodman M.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8919-8924Crossref PubMed Scopus (487) Google Scholar). that polV and RecA promote TLS in the of SSB were used K. K. C. Woodgate R. Cox M.M. Goodman M.F. Mol. Cell. 17: Full Text Full Text PDF PubMed Scopus Google Scholar), RecA well the in were (22Fujii S. Gasser V. Fuchs R.P. J. Mol. Biol. 2004; 341: 405-417Crossref PubMed Scopus (60) Google Scholar). under conditions that are with the with to DNA and RecA SSB is essential for TLS by polV (21Pham P. Bertram J.G. O'Donnell M. Woodgate R. Goodman M.F. Nature. 2001; 409: 366-370Crossref PubMed Scopus (110) Google Scholar, N.B. Arad G. A. Livneh Z. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. Fuchs R.P. EMBO J. 25: PubMed Scopus Google Scholar). of SSB are in its in TLS: (a) It is to the accessibility of the primer terminus to proteins, as indicated by the exonuclease protection This is mediated via the ability of SSB to the template DNA at the site of the and the RecA filament promote its This mechanism is by the ability of SSB to strongly ssDNA (15Lohman T.M. Ferrari M.E. Annu. Rev. Biochem. 1994; 63: 527-570Crossref PubMed Scopus (528) Google and by the that the heterologous ssDNA-binding protein had on primer accessibility, as in this This binding to be it not a of the RecA nucleoprotein (b) It interacts with protein in the TLS This mechanism is by the TLS for SSB-113 and by the direct interaction SSB and as by the The study of the SSB-113 mutant was this two functions of SSB in TLS. The binding of SSB-113 to ssDNA is to that of as J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the that it wild-type ssDNA binding, it is deficient in supporting polV-catalyzed TLS in The SSB-113 located in the C terminus of was to the binding of SSB to proteins J. Curth U. Urbanke C. Biol. Chem. 2000; 381: 183-192Crossref PubMed Scopus (99) Google Scholar, 19Kelman Z. Yuzhakov A. Andjelkovic J. O'Donnell M. EMBO J. 1998; 17: 2436-2449Crossref PubMed Scopus (155) Google Scholar, 20Witte G. Urbanke C. Curth U. Nucleic Acids Res. 2004; 31: 4434-4440Crossref Scopus (88) Google Scholar, K. R.D. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, P. U. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). to the in binding to its S. K. T.M. G. Sci. 2004; 13: PubMed Scopus Google Scholar, R. G. Urbanke C. Curth U. Nucleic Acids Res. PubMed Scopus Google Scholar). The direct interaction of SSB with via its UmuC in the recruitment of polV to the primer-template at the site of a This interaction be by the proteins, which are for TLS S. A. Fuchs R.P. EMBO J. 25: PubMed Scopus Google Scholar, R.D. J. Mol. PubMed Scopus Google Scholar, J. 1998; PubMed Google Scholar, Res. PubMed Scopus Google Scholar). The here that SSB and RecA are roles the response to DNA damage that blocks the replication an it this stage the ssDNA in the is to which an essential role in in DNA, and it for replication the of the ssDNA a to its it to This is by the RecA protein, which is to in the and the ssDNA with a This RecA nucleoprotein filament to the SOS response under the promotes for TLS to SSB via its ability to interacts with polV the initiation of TLS. It was that the RecA filament in DNA to promote TLS K. Cox M.M. Woodgate R. Goodman M.F. Nature. PubMed Scopus Google Scholar). study was in the of the the importance of SSB in TLS (21Pham P. Bertram J.G. O'Donnell M. Woodgate R. Goodman M.F. Nature. 2001; 409: 366-370Crossref PubMed Scopus (110) Google Scholar). results not the results by Goodman and but a that not in to our the RecA filament is formed replication at a as described and a and for It promotes the autocleavage of LexA to of the SOS and it promotes recombination as an tolerance UmuC and UmuD are UmuD is to UmuD′ and polV is SSB binds to the primer-template-lesion polV via its interaction with and is by the interaction UmuD′ and RecA This reaction be by the proteins S. A. Fuchs R.P. EMBO J. 25: PubMed Scopus Google Scholar). The gene is and and defective and in of the SOS response Mol. 1981; PubMed Scopus Google Scholar, Mol. 1981; PubMed Scopus (33) Google Scholar, J. PubMed Google Scholar). It was that the in these is in the of the SOS response Mol. PubMed Scopus Google Scholar). this not also a direct role in as In SSB polV-catalyzed TLS by two which are by the (a) ssDNA binding increases the accessibility of 3′ primer terminus, binding of This of the RecA filament (2Goodman M.F. Trends Biochem. Sci. 2000; 25: 189-195Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). (b) direct interaction with mediated by its C terminus, recruit polV to the DNA. We I. Lehman of for the of the protein and O'Donnell for the of the complex and with