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• W2000842220 abstract "The MucA and MucB proteins are plasmid-encoded homologues of the Escherichia coli UmuD and UmuC proteins, respectively. These proteins are required for SOS mutagenesis, although their mechanism of action is unknown. By using the yeast two-hybrid system we have discovered that MucB interacts with SSB, the single strand DNA binding protein (SSB) of E. coli. To examine the interaction at the protein level, the MucA, MucA′, and MucB proteins were overproduced, purified in denatured state, and refolded. Purified MucA and MucA′ each formed homodimers, whereas MucB was a monomer under native conditions. RecA promoted the cleavage of MucA to MucA′, and MucB was found to bind single-stranded DNA (ssDNA), similarly to the properties of the homologous UmuD and UmuC proteins. Purified MucB caused a shift in the migration of SSB in a sucrose density gradient, consistent with an interaction between these proteins. Addition of MucB to SSB-coated ssDNA caused increased electrophoretic mobility of the nucleoprotein complex and increased staining of the DNA by ethidium bromide. Analysis of radiolabeled SSB in the complexes revealed that only a marginal release of SSB occurred upon addition of MucB. These results suggest that MucB induces a major conformational change in the SSB·ssDNA complex but does not promote massive release of SSB from the DNA. The interaction with SSB might be related to the role of MucB in SOS-regulated mutagenesis. The MucA and MucB proteins are plasmid-encoded homologues of the Escherichia coli UmuD and UmuC proteins, respectively. These proteins are required for SOS mutagenesis, although their mechanism of action is unknown. By using the yeast two-hybrid system we have discovered that MucB interacts with SSB, the single strand DNA binding protein (SSB) of E. coli. To examine the interaction at the protein level, the MucA, MucA′, and MucB proteins were overproduced, purified in denatured state, and refolded. Purified MucA and MucA′ each formed homodimers, whereas MucB was a monomer under native conditions. RecA promoted the cleavage of MucA to MucA′, and MucB was found to bind single-stranded DNA (ssDNA), similarly to the properties of the homologous UmuD and UmuC proteins. Purified MucB caused a shift in the migration of SSB in a sucrose density gradient, consistent with an interaction between these proteins. Addition of MucB to SSB-coated ssDNA caused increased electrophoretic mobility of the nucleoprotein complex and increased staining of the DNA by ethidium bromide. Analysis of radiolabeled SSB in the complexes revealed that only a marginal release of SSB occurred upon addition of MucB. These results suggest that MucB induces a major conformational change in the SSB·ssDNA complex but does not promote massive release of SSB from the DNA. The interaction with SSB might be related to the role of MucB in SOS-regulated mutagenesis. UV mutagenesis in Escherichia coli is a regulated process, controlled by the SOS stress response through its two global regulators, RecA and LexA. The mechanism underlying this process is trans-lesion replication by a DNA polymerase, most likely DNA polymerase III (for reviews see Refs. 1Walker G.C. Microbiol. Rev. 1984; 48: 60-93Crossref PubMed Google Scholar, 2Livneh Z. Cohen-Fix O. Skaliter R. Elizur T. CRC Crit. Rev. Biochem. Mol. Biol. 1993; 28: 465-513Crossref PubMed Scopus (100) Google Scholar, 3Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995Google Scholar). This process requires specifically two SOS-induced proteins, UmuD and UmuC (4Kato T. Shinoura Y. Mol. Gen. Genet. 1977; 156: 121-131Crossref PubMed Scopus (436) Google Scholar, 5Elledge S.J. Walker G.C. J. Mol. Biol. 1983; 164: 175-192Crossref PubMed Scopus (166) Google Scholar, 6Shinagawa H. Kato T. Ise T. Makino K. Nakata A. Gene (Amst .). 1983; 23: 167-174Crossref PubMed Scopus (153) Google Scholar), whose mechanism of action is unknown. A prevailing hypothesis is that UmuD′, the active form of UmuD (7Shinagawa H. Iwasaki H. Kato T. Nakata A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1806-1810Crossref PubMed Scopus (269) Google Scholar, 8Burckhardt S.E. Woodgate R. Scheuermann R.H. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1811-1815Crossref PubMed Scopus (274) Google Scholar, 9Nohmi T. Battista J.R. Dodson L.A. Walker G.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1816-1820Crossref PubMed Scopus (314) Google Scholar), along with UmuC are required to assist the DNA polymerase in replicating the damaged site (10Rajagopalan M. Lu C. Woodgate R. O'Donnell M. Goodman M. Echols M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10777-10781Crossref PubMed Scopus (182) Google Scholar). However, the nature of this assistance is not clear because purified DNA polymerases can bypass DNA lesions unassisted (11Kunkel T.A. Shearman C.W. Loeb L.A. Nature. 1981; 291: 349-351Crossref PubMed Scopus (44) Google Scholar, 12Schaaper R.M. Kunkel T.A. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 487-491Crossref PubMed Scopus (270) Google Scholar, 13Sagher D. Strauss B. Biochemistry. 1983; 22: 4518-4526Crossref PubMed Scopus (295) Google Scholar, 14Strauss B.S. Cancer Surv. 1985; 4: 493-516PubMed Google Scholar, 15Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4599-4603Crossref PubMed Scopus (40) Google Scholar, 16Hevroni D. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5046-5050Crossref PubMed Scopus (51) Google Scholar, 17Taylor J.S. O'Day C.L. Biochemistry. 1990; 29: 1624-1632Crossref PubMed Scopus (68) Google Scholar, 18Paz-Elizur T. Takeshita M. Goodman M. O'Donnell M. Livneh Z. J. Biol. Chem. 1996; 271: 24662-24669Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 19Paz-Elizur T. Takeshita M. Livneh Z. Biochemistry. 1997; 36: 1766-1773Crossref PubMed Scopus (46) Google Scholar). Moreover, in an in vitro system for UV mutagenesis carried out with crude protein extracts (20Cohen-Fix O. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3300-3304Crossref PubMed Scopus (32) Google Scholar, 21Cohen-Fix O. Livneh Z. J. Biol. Chem. 1994; 269: 4953-4958Abstract Full Text PDF PubMed Google Scholar) or with purified proteins (22Tomer G. Cohen-Fix O. O'Donnell M. Goodman M. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1376-1380Crossref PubMed Scopus (10) Google Scholar), we have found that UV mutations were effectively produced in the absence of UmuD′ and UmuC.Homologues of UmuD and UmuC have been identified in other bacteria, and some of them are encoded by conjugational plasmids (23Woodgate R. Levine A.S. Cancer Surv. 1996; 28: 117-140PubMed Google Scholar, 24Szekeres E.J. Woodgate R. Lawrence C.W. J. Bacteriol. 1996; 178: 2559-2563Crossref PubMed Google Scholar). The most well-studied of these are the mucA and mucBgenes, encoding homologues of UmuD and UmuC, respectively (25Perry K.L. Walker G.C. Nature. 1982; 300: 278-281Crossref PubMed Scopus (115) Google Scholar, 26Perry K.L. Elledge S.J. Mitchell B.B. Marsh L. Walker G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4331-4335Crossref PubMed Scopus (170) Google Scholar). An approach that has proven useful in the study of many proteins is to examine their interactions with other proteins. Employing this approach we present here in vivo and in vitro data that show, for the first time, that MucB interacts with SSB 1The abbreviations used are: SSB, single strand DNA binding protein; ssDNA, single-stranded DNA; DTT, dithiothreitol; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; ATPγS, adenosine 5′-O-(thiotriphosphate). 1The abbreviations used are: SSB, single strand DNA binding protein; ssDNA, single-stranded DNA; DTT, dithiothreitol; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; ATPγS, adenosine 5′-O-(thiotriphosphate). and greatly changes the structure of the SSB·ssDNA complex.DISCUSSIONThe yeast two-hybrid system had proven useful in identifying interactions of the Muc proteins. It was previously shown that the UmuD′ and UmuD proteins form both homo- and heterodimers when assayed biochemically (35Woodgate R. Rajagopalan M. Lu C. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7301-7305Crossref PubMed Scopus (185) Google Scholar, 36Battista J.R. Ohta T. Nohmi T. Sun W. Walker G.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7190-7194Crossref PubMed Scopus (103) Google Scholar, 45Jonczyk P. Nowicka A. J. Bacteriol. 1996; 178: 2580-2585Crossref PubMed Google Scholar) and in the yeast two-hybrid system (45Jonczyk P. Nowicka A. J. Bacteriol. 1996; 178: 2580-2585Crossref PubMed Google Scholar). Here we show that MucA′ and MucA behave similarly and exhibit both self-interactions and an interaction with each other. Each of these proteins was found to interact also with MucB. The C-terminal 30 amino acids of MucB were critical in mediating this interaction, since their deletion abolished the interaction of MucB with both MucA and MucA′. The C terminus of the UmuC protein had been previously reported to be essential for UV mutagenesis (46Woodgate R. Singh M. Kulaeva O.I. Frank E.G. Levine A.S. Koch W.H. J. Bacteriol. 1994; 176: 5011-5021Crossref PubMed Google Scholar). Based on the homology between UmuC and MucB, our results suggest that the interaction between MucB and MucA′ is crucial for the activity of these proteins in UV mutagenesis, consistent with the current view that the active species in mutagenesis is a complex of MucA′ and MucB (or UmuD′ and UmuC) (reviewed in Ref.47Walker G.C. Trends Biochem. Sci. 1995; 20: 416-420Abstract Full Text PDF PubMed Scopus (91) Google Scholar). Interestingly, it has been reported that UmuC interacts with UmuD′, but not with UmuD, in the yeast two-hybrid system (45Jonczyk P. Nowicka A. J. Bacteriol. 1996; 178: 2580-2585Crossref PubMed Google Scholar). In our case, MucB was found to interact both with MucA and MucA′. At this stage it is not clear whether these differences are real or whether they reflect differences in the assay systems.The interaction between MucA and RecA as revealed in the two-hybrid system was expected based on the finding that RecA promotes the cleavage of MucA to MucA′ (37Shiba T. Iwasaki H. Nakata A. Shinagawa H. Mol. Gen. Genet. 1990; 224: 169-176Crossref PubMed Scopus (19) Google Scholar, 38Hauser J. Levine A.S. Ennis D.G. Chumakov K.M. Woodgate R. J. Bacteriol. 1992; 174: 6844-6851Crossref PubMed Google Scholar), similarly to the cleavage of UmuD to UmuD′ (7Shinagawa H. Iwasaki H. Kato T. Nakata A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1806-1810Crossref PubMed Scopus (269) Google Scholar, 8Burckhardt S.E. Woodgate R. Scheuermann R.H. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1811-1815Crossref PubMed Scopus (274) Google Scholar, 9Nohmi T. Battista J.R. Dodson L.A. Walker G.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1816-1820Crossref PubMed Scopus (314) Google Scholar). Interactions between RecA and MucA′ or UmuD′ were proposed previously based on chemical cross-linking experiments (39Frank 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). Taken together with our in vivo results, it seems plausible that the MucA/A′-RecA interactions fulfill a role additional to mediating the cleavage of MucA, possibly the recruitment of MucA′ to DNA, as previously suggested (39Frank 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). We found no interaction between MucB and RecA. A related binding interaction was the retention of UmuC on activated RecA immobilized on a column, when an extract of E. coli cells was passed through the column (48Freitag N. McEntee K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8363-8367Crossref PubMed Scopus (35) Google Scholar). However, in this reported case, since an extract (rather than purified proteins) was used, the possibility that a third protein had mediated the interaction between RecA and UmuC hasn't been ruled out.SSB, reported here to interact with MucB, is an essential protein, which is involved in DNA replication, recombination, and repair (49Chase J.W. Williams K.R. Annu. Rev. Biochem. 1986; 55: 103-136Crossref PubMed Scopus (444) Google Scholar, 50Kornberg A. Baker T. DNA Replication. W. H. Freeman & Co., New York1991Google Scholar, 51Meyer R.R. Laine P.S. Microbiol. Rev. 1990; 54: 342-380Crossref PubMed Google Scholar). It is a homotetramer that binds ssDNA specifically and in a cooperative manner. Each SSB monomer contains a DNA-binding site interacting with 16 nucleotides. The extent of DNA bound by SSB strongly depends on salt and Mg2+ concentrations. With the increase in their concentration, the number of subunits that interact with DNA increases from 2 (SSB35 mode) to 4 (SSB65 mode). Electron microscopic analysis revealed that at low SSB to DNA ratio, a beaded structure is observed, with the DNA wrapped around beads of single or double tetramers of SSB, leading to a reduction in the contour length of DNA. This binding is of limited cooperativity and represents most likely the SSB65 mode. At higher SSB to DNA ratios, the binding to DNA is cooperative leading to the formation of a smooth SSB-DNA filament, where SSB is presumably in the 35 mode (reviewed in Refs. 44Lohman T.M. Bujalowski W. Overman L.B. Trends Biochem. Sci. 1988; 13: 250-255PubMed Google Scholar, 50Kornberg A. Baker T. DNA Replication. W. H. Freeman & Co., New York1991Google Scholar, and 52Lohman T.M. Ferrari M.E. Annu. Rev. Biochem. 1994; 63: 527-570Crossref PubMed Scopus (523) Google Scholar).Our results suggest that the interaction with MucB affects the cooperative mode of SSB binding (presumably the 35-mode), which is the initial binding state of the SSB under our conditions. The exact nature of the MucB-SSB interaction is not clear yet, but it causes a major change in the SSB·ssDNA complex, without causing massive release of the SSB from DNA. This is evident from the increase in the mobility of the nucleoprotein complex and its increased staining with ethidium bromide. The quantitation of radiolabeled SSB is accurate within 10–15%. Thus, while not causing a massive displacement from DNA, it is possible that MucB does replace some SSB molecules. This, however, must have a major effect on the conformation of the DNA, as evident from its increased mobility. Several attempts to examine whether the nucleoprotein complex contains the MucB protein, using anti-MucB antibodies, were unsuccessful. At this point it is not clear whether this is a technical problem or whether MucB is released after rearranging the SSB·ssDNA complex. The increased staining can be explained by at least the following two mechanisms: 1) an increase in the accessibility of the DNA bases to ethidium bromide and 2) a change in the stacking of the DNA bases, which enables better intercalation of the dye. Noteworthy, although MucB bound a ssDNA 40 nucleotides long, we found no effect of MucB alone, or in combination with MucA or MucA′, on the migration of naked M13mp8 ssDNA in agarose gel electrophoresis. The reasons for this result are not clear yet.What are the consequences of the changes in the structure of the nucleoprotein complex caused by MucB? It is possible that such changes allow easier bypass of DNA lesions by a DNA polymerase or else they provide binding sites for other proteins required for the bypass reaction. A natural candidate for being involved in this reaction is the RecA protein. It has been shown to compete with SSB for binding to ssDNA and under certain conditions can form a ternary SSB·RecA·ssDNA complex (reviewed in Refs. 53Roca A.I. Cox M.M. CRC Crit. Rev. Biochem. Mol. Biol. 1990; 25: 415-456Crossref PubMed Scopus (363) Google Scholar and 54Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar). MucB interacted with SSB but not with RecA (Table IV). On the other hand, MucA′ did interact with RecA. This may mean that a MucA′B complex interacts with both RecA and SSB. The interactions of MucB with SSB and with MucA′ must occur via different regions of the protein. The C terminus of MucB is involved in the interaction with MucA/A′, whereas an additional domain is involved in the interaction with SSB. Thus, a putative 4-protein RecA-MucA′-MucB-SSB complex can be imagined, in which RecA is bound to MucA′B via MucA′, and SSB is bound via MucB. So far there is no evidence for such a complex. An alternative possibility is that the various interactions appear transiently during the mutagenic bypass reaction.One of the paradoxes in the field of SOS mutagenesis is that in vivo bypass of DNA lesions requires the UmuD′ and UmuC proteins (or their homologues) (reviewed in Ref. 3Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995Google Scholar), whereas in vitrobypass of blocking lesions (2Livneh Z. Cohen-Fix O. Skaliter R. Elizur T. CRC Crit. Rev. Biochem. Mol. Biol. 1993; 28: 465-513Crossref PubMed Scopus (100) Google Scholar, 11Kunkel T.A. Shearman C.W. Loeb L.A. Nature. 1981; 291: 349-351Crossref PubMed Scopus (44) Google Scholar, 12Schaaper R.M. Kunkel T.A. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 487-491Crossref PubMed Scopus (270) Google Scholar, 13Sagher D. Strauss B. Biochemistry. 1983; 22: 4518-4526Crossref PubMed Scopus (295) Google Scholar, 14Strauss B.S. Cancer Surv. 1985; 4: 493-516PubMed Google Scholar, 15Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4599-4603Crossref PubMed Scopus (40) Google Scholar, 16Hevroni D. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5046-5050Crossref PubMed Scopus (51) Google Scholar, 17Taylor J.S. O'Day C.L. Biochemistry. 1990; 29: 1624-1632Crossref PubMed Scopus (68) Google Scholar, 18Paz-Elizur T. Takeshita M. Goodman M. O'Donnell M. Livneh Z. J. Biol. Chem. 1996; 271: 24662-24669Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 19Paz-Elizur T. Takeshita M. Livneh Z. Biochemistry. 1997; 36: 1766-1773Crossref PubMed Scopus (46) Google Scholar) and in vitro UV mutagenesis (20Cohen-Fix O. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3300-3304Crossref PubMed Scopus (32) Google Scholar, 21Cohen-Fix O. Livneh Z. J. Biol. Chem. 1994; 269: 4953-4958Abstract Full Text PDF PubMed Google Scholar, 22Tomer G. Cohen-Fix O. O'Donnell M. Goodman M. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1376-1380Crossref PubMed Scopus (10) Google Scholar) can occur without these proteins. The report onumuC-independent UV mutagenesis in phage S13 (55Tessman I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6614-6618Crossref PubMed Scopus (38) Google Scholar) and the finding that umuC-independent UV mutagenesis is observed when a screening rather than selection procedure is used (56Christensen J.R. LeClerc J.E. Valone Tata P. Christensen R.B. Lawrence C.W. J. Mol. Biol. 1988; 203: 635-641Crossref PubMed Scopus (21) Google Scholar) support the view that UmuD′C stimulates the mutagenic reaction, rather than being absolutely required. An obvious difference between the in vivo and in vitro situations is that in the former there exist many more proteins that may affect bypass. One such family includes DNA damage binding proteins, which usually function in error-free DNA repair (3Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995Google Scholar). We have recently shown that DNA damage binding proteins regulate induced mutagenesis via a mechanism that does not involve the removal of DNA damage. We found that DNA damage binding proteins directly inhibit trans-lesion replication by binding to lesions present on ssDNA (57Paz-Elizur T. Barak Y. Livneh Z. J. Biol. Chem. 1997; 272: 28906-28911Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). The current study points toward SSB as another candidate that might affect SOS mutagenesis. in vivostudies have shown that E. coli strains carrying the ssb1 mutation, encoding a temperature-sensitive SSB, had reduced UV mutagenesis at the non-permissive temperature (58Lieberman H.B. Witkin E.M. Mol. Gen. Genet. 1981; 183: 348-355Crossref PubMed Scopus (28) Google Scholar, 59Lieberman H.B. Witkin E.M. Mol. Gen. Genet. 1983; 190: 92-100Crossref PubMed Scopus (51) Google Scholar). However, this was attributed to the inability of the mutant to fully induce the SOS response. Of course, this analysis does not rule out the possibility of a direct involvement of SSB in the mutagenic reaction. We have previously reported that SSB facilitates unassisted bypass of UV lesions by DNA polymerase III holoenzyme (29Livneh Z. J. Biol. Chem. 1986; 261: 9526-9533Abstract Full Text PDF PubMed Google Scholar). It is possible that the MucB·SSB complex increases bypass, especially when additional proteins are present. The exact mechanism of the involvement of SSB in SOS UV mutagenesis needs further biochemical and in vivo investigations. UV mutagenesis in Escherichia coli is a regulated process, controlled by the SOS stress response through its two global regulators, RecA and LexA. The mechanism underlying this process is trans-lesion replication by a DNA polymerase, most likely DNA polymerase III (for reviews see Refs. 1Walker G.C. Microbiol. Rev. 1984; 48: 60-93Crossref PubMed Google Scholar, 2Livneh Z. Cohen-Fix O. Skaliter R. Elizur T. CRC Crit. Rev. Biochem. Mol. Biol. 1993; 28: 465-513Crossref PubMed Scopus (100) Google Scholar, 3Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995Google Scholar). This process requires specifically two SOS-induced proteins, UmuD and UmuC (4Kato T. Shinoura Y. Mol. Gen. Genet. 1977; 156: 121-131Crossref PubMed Scopus (436) Google Scholar, 5Elledge S.J. Walker G.C. J. Mol. Biol. 1983; 164: 175-192Crossref PubMed Scopus (166) Google Scholar, 6Shinagawa H. Kato T. Ise T. Makino K. Nakata A. Gene (Amst .). 1983; 23: 167-174Crossref PubMed Scopus (153) Google Scholar), whose mechanism of action is unknown. A prevailing hypothesis is that UmuD′, the active form of UmuD (7Shinagawa H. Iwasaki H. Kato T. Nakata A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1806-1810Crossref PubMed Scopus (269) Google Scholar, 8Burckhardt S.E. Woodgate R. Scheuermann R.H. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1811-1815Crossref PubMed Scopus (274) Google Scholar, 9Nohmi T. Battista J.R. Dodson L.A. Walker G.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1816-1820Crossref PubMed Scopus (314) Google Scholar), along with UmuC are required to assist the DNA polymerase in replicating the damaged site (10Rajagopalan M. Lu C. Woodgate R. O'Donnell M. Goodman M. Echols M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10777-10781Crossref PubMed Scopus (182) Google Scholar). However, the nature of this assistance is not clear because purified DNA polymerases can bypass DNA lesions unassisted (11Kunkel T.A. Shearman C.W. Loeb L.A. Nature. 1981; 291: 349-351Crossref PubMed Scopus (44) Google Scholar, 12Schaaper R.M. Kunkel T.A. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 487-491Crossref PubMed Scopus (270) Google Scholar, 13Sagher D. Strauss B. Biochemistry. 1983; 22: 4518-4526Crossref PubMed Scopus (295) Google Scholar, 14Strauss B.S. Cancer Surv. 1985; 4: 493-516PubMed Google Scholar, 15Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4599-4603Crossref PubMed Scopus (40) Google Scholar, 16Hevroni D. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5046-5050Crossref PubMed Scopus (51) Google Scholar, 17Taylor J.S. O'Day C.L. Biochemistry. 1990; 29: 1624-1632Crossref PubMed Scopus (68) Google Scholar, 18Paz-Elizur T. Takeshita M. Goodman M. O'Donnell M. Livneh Z. J. Biol. Chem. 1996; 271: 24662-24669Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 19Paz-Elizur T. Takeshita M. Livneh Z. Biochemistry. 1997; 36: 1766-1773Crossref PubMed Scopus (46) Google Scholar). Moreover, in an in vitro system for UV mutagenesis carried out with crude protein extracts (20Cohen-Fix O. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3300-3304Crossref PubMed Scopus (32) Google Scholar, 21Cohen-Fix O. Livneh Z. J. Biol. Chem. 1994; 269: 4953-4958Abstract Full Text PDF PubMed Google Scholar) or with purified proteins (22Tomer G. Cohen-Fix O. O'Donnell M. Goodman M. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1376-1380Crossref PubMed Scopus (10) Google Scholar), we have found that UV mutations were effectively produced in the absence of UmuD′ and UmuC. Homologues of UmuD and UmuC have been identified in other bacteria, and some of them are encoded by conjugational plasmids (23Woodgate R. Levine A.S. Cancer Surv. 1996; 28: 117-140PubMed Google Scholar, 24Szekeres E.J. Woodgate R. Lawrence C.W. J. Bacteriol. 1996; 178: 2559-2563Crossref PubMed Google Scholar). The most well-studied of these are the mucA and mucBgenes, encoding homologues of UmuD and UmuC, respectively (25Perry K.L. Walker G.C. Nature. 1982; 300: 278-281Crossref PubMed Scopus (115) Google Scholar, 26Perry K.L. Elledge S.J. Mitchell B.B. Marsh L. Walker G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4331-4335Crossref PubMed Scopus (170) Google Scholar). An approach that has proven useful in the study of many proteins is to examine their interactions with other proteins. Employing this approach we present here in vivo and in vitro data that show, for the first time, that MucB interacts with SSB 1The abbreviations used are: SSB, single strand DNA binding protein; ssDNA, single-stranded DNA; DTT, dithiothreitol; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; ATPγS, adenosine 5′-O-(thiotriphosphate). 1The abbreviations used are: SSB, single strand DNA binding protein; ssDNA, single-stranded DNA; DTT, dithiothreitol; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; ATPγS, adenosine 5′-O-(thiotriphosphate). and greatly changes the structure of the SSB·ssDNA complex. DISCUSSIONThe yeast two-hybrid system had proven useful in identifying interactions of the Muc proteins. It was previously shown that the UmuD′ and UmuD proteins form both homo- and heterodimers when assayed biochemically (35Woodgate R. Rajagopalan M. Lu C. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7301-7305Crossref PubMed Scopus (185) Google Scholar, 36Battista J.R. Ohta T. Nohmi T. Sun W. Walker G.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7190-7194Crossref PubMed Scopus (103) Google Scholar, 45Jonczyk P. Nowicka A. J. Bacteriol. 1996; 178: 2580-2585Crossref PubMed Google Scholar) and in the yeast two-hybrid system (45Jonczyk P. Nowicka A. J. Bacteriol. 1996; 178: 2580-2585Crossref PubMed Google Scholar). Here we show that MucA′ and MucA behave similarly and exhibit both self-interactions and an interaction with each other. Each of these proteins was found to interact also with MucB. The C-terminal 30 amino acids of MucB were critical in mediating this interaction, since their deletion abolished the interaction of MucB with both MucA and MucA′. The C terminus of the UmuC protein had been previously reported to be essential for UV mutagenesis (46Woodgate R. Singh M. Kulaeva O.I. Frank E.G. Levine A.S. Koch W.H. J. Bacteriol. 1994; 176: 5011-5021Crossref PubMed Google Scholar). Based on the homology between UmuC and MucB, our results suggest that the interaction between MucB and MucA′ is crucial for the activity of these proteins in UV mutagenesis, consistent with the current view that the active species in mutagenesis is a complex of MucA′ and MucB (or UmuD′ and UmuC) (reviewed in Ref.47Walker G.C. Trends Biochem. Sci. 1995; 20: 416-420Abstract Full Text PDF PubMed Scopus (91) Google Scholar). Interestingly, it has been reported that UmuC interacts with UmuD′, but not with UmuD, in the yeast two-hybrid system (45Jonczyk P. Nowicka A. J. Bacteriol. 1996; 178: 2580-2585Crossref PubMed Google Scholar). In our case, MucB was found to interact both with MucA and MucA′. At this stage it is not clear whether these differences are real or whether they reflect differences in the assay systems.The interaction between MucA and RecA as revealed in the two-hybrid system was expected based on the finding that RecA promotes the cleavage of MucA to MucA′ (37Shiba T. Iwasaki H. Nakata A. Shinagawa H. Mol. Gen. Genet. 1990; 224: 169-176Crossref PubMed Scopus (19) Google Scholar, 38Hauser J. Levine A.S. Ennis D.G. Chumakov K.M. Woodgate R. J. Bacteriol. 1992; 174: 6844-6851Crossref PubMed Google Scholar), similarly to the cleavage of UmuD to UmuD′ (7Shinagawa H. Iwasaki H. Kato T. Nakata A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1806-1810Crossref PubMed Scopus (269) Google Scholar, 8Burckhardt S.E. Woodgate R. Scheuermann R.H. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1811-1815Crossref PubMed Scopus (274) Google Scholar, 9Nohmi T. Battista J.R. Dodson L.A. Walker G.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1816-1820Crossref PubMed Scopus (314) Google Scholar). Interactions between RecA and MucA′ or UmuD′ were proposed previously based on chemical cross-linking experiments (39Frank 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). Taken together with our in vivo results, it seems plausible that the MucA/A′-RecA interactions fulfill a role additional to mediating the cleavage of MucA, possibly the recruitment of MucA′ to DNA, as previously suggested (39Frank E.G. Hauser J. Levine A.S. Woodgate R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8" @default.
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• W2000842220 title "The Mutagenesis Protein MucB Interacts with Single Strand DNA Binding Protein and Induces a Major Conformational Change in Its Complex with Single-stranded DNA" @default.
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