Proc. Natl. Acad. Sci. USA Vol. 89, pp. 10777-10781, November 1992 Biochemistry Activity of the purified mutagenesis proteins UmuC, UmuD', and RecA in replicative bypass of an abasic DNA lesion by DNA polymerase III (SOS respon/fdelity of DNA repfcatlon/umutatlo) MALINI RAJAGOPALANt, CHI Lut, ROGER WOODGATEtt, MIKE O'DONNELL§, MYRON F. GOODMAN$, AND HARRISON ECHOLSt tDivision of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720; 1Department of Microbiology, Cornell University Medical College, New York, NY 10021; and IDepartment of Biological Science, University of Southern California, Los Angeles, CA 90089 Contributed by Harrison Echols, August 17, 1992

ABSTRACT The introduction of a replication-inhibiting SOS mutagenesis (C. Bonner, S. Creighton and M.F.G., lesion Into the DNA ofEscherichia colU generates the Induced, unpublished work). multigene SOS response. One component of the SOS response Together, the studies noted above give clear indications is a marked increase in mutation rate, de t on RecA that SOS mutagenesis is a consequence ofreplicative bypass protein and the induced muta p us UmuC and of DNA lesions mediated by a damage-localized nucleopro- UmuD. A variety of previous indirect ap es have Indi- tein complex involving RecA, UmuC-UmuD', and pol III-a cated that SOS mutagenesi results from replicative bypass of "mutasome" (14, 17). However, direct evidence for such a the DNA lesion by DNA polymerase (pol ) me in pathway has been lacking in the absence of a defined bio- a reaction mediated by RecA, UmuC, and a prcsd form of chemical system. In the work reported here, we have used UmuD termed UmuD'. To study the bc is of SOS purified proteins to demonstrate replicative bypass of an mutagenesis, we have recostutd replicative bypass with a abasic lesion in a reaction requiring UmuC, UmuD', and defined in vitro system cotg purified proteIns and a DNA RecA. Thus we have concluded that the UmuC-UmuD' substrate with a singe abasc DNA lesion. The replicative complex and RecA act to rescue an otherwise stalled pol III bypass reaction requires po0 m1, UmuC, UmuD', and RecA. holoenzyme at a replication-blocking DNA lesion. The nonprocessed UmuD protein does not replace UmuD' but inhibits the bypass activity of UmuD', perhaps by sequestering MATERIALS AND METHODS UmuD' In a heterodimer. Our experiments demonstrate di- rectly that the UmuC-UmuD' complex and RecA act to rescue Materials. T4 polynucleotide kinase, Fsp I restriction en- an otherwise stalled po1 II holoenzyme at a replication- zyme, DNA ligase, and 4X174 single-stranded DNA (ssDNA) blocking DNA lesion. were obtained from New England Biolabs; ultrapure ATP and deoxynucleoside triphosphates and DNA polymerase I (Kle- A population of Escherichia coli bacteria reacts to a repli- now fragment) were from Pharmacia; [y32P]ATP (3000 Ci/ cation-blocking DNA lesion by inducing the multigene SOS mmol; 1 Ci = 37 GBq) was from Amersham; and polyethylene response (1-4). A remarkable and intensely studied aspect of glycol (PEG) 6000 was from Sigma. Sequenase was obtained the SOS response is an induced mutagenic pathway that from United States Biochemical. The oligonucleotide template depends on the RecA protein and the UmuC and UmuD with a specific abasic site was prepard as described (27). proteins (4-14). SOS mutagenesis is regulated by at least two Other oligonucleotides used were either from Appligene sequential reactions. First, RecA protein is activated by (Pleasanton, CA) or from the Microchemical Facility at the DNA damage to mediate the proteolytic cleavage of the University of California, Berkeley. Purified proteins used in LexA repressor for SOS-controlled genes, including umuC the assays were prepared as described: a (28), as (29), y and umuD (2, 13). In a second RecA-dependent proteolytic complex (30), pol III* (31), DNA polymerase 11 (32), RecA step, UmuD is processed to UmuD', the active agent in (19), UmuC (17), UmuD (16), and UmuD' (17). The , protein mutagenesis (9, 15, 16). Based on direct evidence for a was a gift from Arthur Kornberg (Stanford University). physical interaction, the UmuC-UmuD' complex is pre- DNA Template Construction. The DNA template used in sumed to be the functional unit for mutagenesis (17). Genetic the replicative bypass assays was a 5.4-kilobase (kb) linear experiments have indicated that RecA probably has a third, ssDNA with an abasic lesion located 30 bases from the 5' end. more direct role in SOS mutagenesis, in addition to the two Circular 4X174 ssDNA was linearized at a unique site by regulatory functions (9-12). annealing a 20-mer oligonucleotide to the ssDNA and cutting Based on the available evidence, the pathway for SOS with Fsp I. The 3' end ofthe linear ssDNA was ligated to the mutagenesis has been presumed to involve replication past 60-mer containing the abasic lesion. The ligation step was the lesion by DNA polymerase in an altered low-fidelity made possible by annealing a 35-mer oligonucleotide which mode mediated by UmuC, UmuD', and RecA (14, 17-19). bridged the linear 4X174 DNA and the 60-mer to create a Genetic and physiological experiments have implicated DNA stretch of double-stranded DNA for ligase action (+5X174/ polymerase III (pol III) in the mutagenic pathway (20, 21). 35-mer/60-mer in ratio 1:2:3). The success of the ligation Genetic studies indicate that DNA polymerase I is not step (>95%) was verified by DNA sequencing. required (22, 23). DNA polymerase II exhibits the interesting Replication Assay. Standard replication reaction mixtures property of SOS induction (24-26); however, deletion and (10 Al) contained 20 mM Tris-HCl (pH 7.5), 8 mM MgC12, 5 insertion mutations in the gene for polymerase II do not alter Abbreviations: pol III, DNA polymerase III; ssDNA, single- stranded DNA. The publication costs ofthis article were defrayed in part by page charge *Present address: Section on Viruses and Cellular Biology, National payment. This article must therefore be hereby marked "advertisement" Institutes ofChild Health and Human Development, National Insti- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tutes of Health, Bethesda, MD 20892.

10777 10778 Biochemistry: Rajagopalan et al. Proc. Natl. Acad. Sci. USA 89 (1992) mM dithiothreitol, 0.1 mM EDTA, 25 mM sodium glutamate, AB 1 mM ATP, 4%6 (vol/vol) glycerol, 40 gg of bovine serum 31 5 ' 5.4kb albumin per ml, and 5% (wt/vol) PEG 6000. The PEG was P2- P1*_ required for an effective replicative bypass reaction. The Ssb, RecA, primed DNA substrate (2.5 nM) was preincubated for 5 min UmuC, UmuD' 5 min at 300C with 3.1 ,M Ssb, 100 nM 13 protein, 3.6 nM ycomplex, 50 nM UmuC, 1.3 ,uM UmuD', and 2.5 AM RecA. Reactions Polymerase, 10 mi were then brought back to ice, and respective polymerases d NTPs 10 min were added. The reconstituted pol III complexes were used 3' at 20 nM and made as described (29). When used, pol III* was _ * _~~~~~~~~I 5' at 12-20 nM. No y complex was added to pol III* reactions. Replication was initiated by the addition of deoxynucleoside PRODUCTS SEPARATED ON DENATURING GELS triphosphates (60 AuM each). Replication was carried out for 1. Replication Block * - 85 b 10 min at 370C, followed by quenching with 20 Al of 20 mM EDTA in 95% formamide. The DNA was then denatured by 2. Mlsincorporatlon *I 86 b heating, and the replicated primer products were separated 3. Bypass *- by electrophoresis in 10%6 polyacrylamide gels containing >86 b urea and were visualized by autoradiography of dried gels. FIG. 1. Assay for translesion replication. The assay used a doubly primed, linear ssDNA substrate (5.4 kb) with a single abasic lesion located 65 nucleotides from the 3' end ofthe radioactive primer RESULTS (P1). Replication products were separated on denaturing acrylamide Assay for the RepUcative Bypass of an Abasic Lesion. To gels. Only replication products from the end-labeled primer (P1) can develop a biochemical system to study replicative bypass of be visualized on autoradiograms of dried gels. DNA lesions, we used an assay system that is highly sensitive to limited bypass. In principle, an oligonucleotide with a with a similar DNA template carrying a cyclobutane pyrim- lesion at a specific site fulfills this need (24,27,33). However, idine dimer, we also obtained bypass of the lesion site by pol assembly of pol III holoenzyme on a primer-template DNA III dependent on UmuC, UmuD', and RecA (M.R., C. requires at least 36 nucleotides from the 3' and the 5' end of Lawrence, and H.E., unpublished work); thus our in vitro the primer (M.O., unpublished data). To circumvent this replicative bypass reaction was not limited to abasic sites. problem, we used a 5.4-kb ssDNA substrate with an abasic The experiments in Fig. 2 were all done with a reconstituted site located 30 nucleotides upstream from the 5' end (Fig. 1). pol III holoenzyme, which carried subunits essential for The 5.4-kb linear ssDNA was replicated from two 20-mer processivity but lacked the e exonuclease subunit ("a holo- oligonucleotide primers. Primer 1 (P1), which anneals to the enzyme") (29). We used this enzyme because the exonu- substrate 65 nucleotides upstream from the abasic site, was clease can limit the very low level of bypass in the absence labeled at the 5' end with ['t-32P]ATP. Replication from the of the mutagenesis proteins (our unpublished data). How- unlabeled primer 2 (P2), located 347 nucleotides downstream ever, as shown below, pol III holoenzyme with exonuclease from the 3' end of the template, was included to overcome also exhibited a bypass reaction in the presence of possible technical complications arising from long regions of UmuC, ssDNA. Only the replication products from P1 can be visu- UmuD', and RecA. alized on autoradiograms of the gels used to separate the + + + + reaction products. We found that Sequenase (T7 DNA poly- tecA + + + merase lacking the editing exonuclease) bypassed the abasic _ _ sites with a limited block (data not shown). We therefore used + - + + Tk this reaction to locate the positions of abasic sites and bypass bands on gels. The expected products from replicating the substrate with the abasic lesion are 85 nucleotides long if there is a replication block, 86 nucleotides long if there is misincorporation opposite the lesion without bypass, and

Optimal bypass in our assay was obtained at a 2:1 molar + + + + + - Rect ratio of RecA to UmuD' (data not shown). Under these + + + + + - tkul + conditions, the UmuD' concentration was about 20-fold + - + + - UtrmuD' greater than that of UmuC. We could not substantially + 1 2 4 - UmiuD increase the concentration of UmuC because ofthe difficulty of obtaining concentrated soluble preparations of UmuC protein (17). The relatively large amount of UmuD' in our assay might be required for effective UmuC-UmuD' complex formation or other relevant protein-protein interactions. < BYPASS Interestingly, in vivo UmuD has been found in a 13-fold molar excess over UmuC (34). To estimate the efficiency of the bypass reaction, we determined the intensity of the bypass bands, normalized to the intensity of the blocked band at the abasic site (measured at a exposure). In the presence of the mutagenesis lower < BI=oc proteins (UmuC, UmuD', and RecA), =5% of the DNA (ABASIC) chains that replicated up to the lesion bypassed the abasic site. A background level of -0.5% bypass was obtained in the absence of any one of the mutagenesis proteins. Bypass replication was not due to more effective replication up to the lesion in the presence of UmuC, UmuD', and RecA. Repli- 1 2 3 4 5 6 7 8 9 1 0 11 12 lesion was slightly less (-85%) under the cation up to the FIG. 3. Effect of UmuD on translesion replication by pol III. bypass conditions (as determined from lower exposures of Standard translesion reactions were performed as for Fig. 2, in the Fig. 2, data not shown). presence of UmuC and RecA, and in the presence or absence of A striking feature of the data is the ladder-like pattern of UmuD' and/or UmuD. Additions to replication reaction mixtures bypass bands (Fig. 2, lanes 5 and 6). The polymerase ap- were as indicated: for UmuD, the notations 1, 2, and 4 refer to the peared to dissociate or stop replicating shortly after bypass- concentration of UmuD relative to UmuD'. ing the lesion instead of continuing processively to the end of the template. In contrast, most of the elongating primer the capacity for replicative bypass by two other forms of chains reached the abasic site. That is, the ratio of the holoenzyme: "ae holoenzyme," reconstituted from purified integrated intensity of the shorter DNA bands to the abasic subunits with e (29); and "cellular holoenzyme," in which the block, 0.1-0.2, was far less than the intensity ratio of the multisubunit pol III* purified directly from E. coli was shorter bypass products to the full-length bypass band, 4-8. combined with f3 subunit (31). All three forms of pol III The laddering effect did not result from an incomplete DNA holoenzyme exhibited bypass of the abasic lesion in a reac- substrate, because T7 DNA polymerase (Sequenase) pro- tion requiring UmuC, UmuD', and RecA (Fig. 4). We have duced full-length products. Further work will be required to not yet analyzed the different reactions for subtle differences distinguish whether this apparent loss of downstream pro- which might address the question ofwhether inhibition ofthe cessivity is a characteristic feature of the bypass reaction exonuclease activity is required for bypass (14). Although occurring in the presence of mutagenesis proteins or specific RecA can inhibit e activity, as measured by exonuclease to the particular assay used. The assay is likely to amplify a loss of processivity because pol III will probably not reas- am- am CHE semble on the short DNA segment downstream from the ++ - + + - + + ecA abasic site. - - + - -+ - - + UTUC Requirement for Processed UmuD' Instead ofUmuD: UmuD + + + tmD' as an Inhibitor. The processing of UmuD protein (15 kDa) to UmuD' (12 kDa) is essential for SOS mutagenesis (9). Be- _ * 1 cause the processing event leaves most of the amino acid residues of UmuD, a requirement for UmuD' instead of . UmuD is a stringent test ofspecificity. Moreover, not only do UmuD and UmuD' form homodimers (17, 35), but UmuD 4iBYPASS also produces a heterodimer with UmuD' (35); this observa- tion, coupled with genetic inferences, has led to the proposal that the heterodimer might be a negative regulator for mu- tagenesis (35). We therefore have studied replicative bypass in the presence of UmuD (Fig. 3). UmuD failed to replace UmuD' in the standard bypass reaction (Fig. 3, lanes 3 and BLOCcK 4). UmuD also inhibited the activity of UmuD', more effec- (ABASIC) tively with excess UmuD (Fig. 3, lanes 5-10). In principle, _ UmuD should eventually be converted to UmuD' in the ~~..Ws presence ofRecA, but this cleavage reaction would not occur efficiently during the time course of our replication reactions 1 23 4 56 7 8 9 (16). From the data of Figs. 2 and 3, we conclude that the three FIG. 4. Translesion replication by a holoenzyme, ae holoen- and RecA-act to- zyme, and cellular holoenzyme (pol III* plus 8 subunit). Standard mutagenesis proteins-UmuC, UmuD', translesion replication reactions were carried out by the three forms gether to facilitate translesion DNA replication, rescuing an of pol III holoenzyme. The a holoenzyme (aHE) and the ar otherwise blocked pol III holoenzyme (a holoenzyme). holoenzyme (aHE) are reconstituted forms of pol III either lacking Replicative Bypass by p0o mI Holoenzyme with Exonudease. or containing the e subunit, respectively. The cellular holoenzyme We began our experiments with a form ofpol III holoenzyme (cHE) is pol III purified directly from E. coli, which carries all of the reconstituted from purified subunits without the e exonu- subunits except 8. All three polymerases were assayed in the clease subunit ("a holoenzyme") (29). We have examined presence of added P subunit. 10780 Biochemistry: Rajagopalan et al. Proc. Nad. Acad Sci. USA 89 (1992)

+ + + PecA - + - + - + + UvnC + - - + + + Upnm

0 I< BYPASS ransiesIion feplicaLuon.n ir-n+irn ,SS.( I{- cir Replication Restart nr i w alaroir1! I~C.-;,-{I;,.mt

A

2 FIG. 5. Failure ofDNA polymerase II to exhibit bypass ofabasic lesions dependent on RecA, UmuC, and UmuD'. Additions to RC J standard replication reaction mixtures were as indicated for lanes PCI cA 1-4. Replication by DNA polymerase II was also carried out in the FIG. 6. Possible mechanisms involved in translesion replication presence of the f3 subunit and y complex subassembly of pol III and replication-restart. A multiprotein complex is assembled at the holoenzyme and Ssb protein (lanes 5-14), with additions as indi- DNA lesion, including UmuC, UmuD', RecA, and pol III holoen- cated. zyme. This nucleoprotein aggregate provides for replication across the lesion with the introduction of mutations. A possible alternative assays (36, 37), RecA clearly must perform some other pathway for error-free replicative bypass is DNA synthesis with the additional function in replicative bypass because RecA is daughter strand as template in a three-stranded complex generated required for bypass by a holoenzyme, which lacks e entirely. by RecA (14, 17). The bypass reaction might also be accomplished A similar conclusion has been derived from in vivo experi- by downstream replication followed by recombinational repair (42). ments with an a holoenzyme generated by mutation (38). Specificity of the Bypass Reaction for pot M. Experiments The experiments reported here have provided a biochemical in vivo have implicated pol III as the target enzyme for SOS demonstration of the principal feature of this model-the mutagenesis (20, 21). To address this requirement in vitro, we concerted action of UmuC, UmuD', and RecA to allow pol I or II would exhibit a III holoenzyme to bypass a DNA lesion. asked whether DNA polymerase Features of the RepUcative Bypass Reaction in Vitro. The bypass reaction facilitated by UmuC, UmuD', and RecA. limited bypass of the abasic lesion is one aspect of our DNA polymerase I was strongly inhibited by RecA but purified protein system that warrants further comment. Al- showed no increased bypass with UmuC and UmuD' (data though the translesion replication is clearly discernible and not shown). DNA polymerase II by itself was even more responds to the genetically defined protein constituents, the strongly inhibited by RecA than was DNA polymerase I (data fraction ofDNA chains that bypass the lesion is only -5% in not shown); however, this problem was overcome by the the presence of the mutagenesis proteins. There are at least presence of the pol III processivity subunits [shown previ- three possible explanations; we suspect that all of them may ously to promote processive DNA replication by DNA poly- be relevant to the problem. (i) The biochemical system has merase II (32, 39)]. The processive polymerase II did not not been extensively optimized for the bypass reaction. Most exhibit a distinct increase in bypass of the abasic site with notably, we have so far been limited by the difficulty in UmuC, UmuD', and RecA (Fig. 5). In some experiments, obtaining UmuC in high concentration (17). (ii) The transle- there was an indication of a marginal increase in bypass by sion mode of replication may be engineered by evolution to the processive polymerase II in the presence of the muta- be inefficient because of the high mutation rate associated genesis proteins. The data for DNA polymerase I and DNA with this pathway of replicative rescue (43). The major polymerase II indicate that the effective function of the cellular pathway to recovery of DNA replication ("replica- mutagenesis proteins is specific for pol III. However, we tion-restart") appears to be nonmutagenic (14, 44, 45); this have not studied the other polymerases under a wide variety pathway depends on RecA but does not require UmuC and of conditions. UmuD' (except with certain RecA mutants) (14, 44, 45). We have suggested (14, 17) that the replication-restart pathway might work by another mode of replicative bypass, involving DISCUSSION a strand-switch copying of an already-replicated daughter Replicative Bypass of DNA Lesions and SOS Mutagenesis. strand (see Fig. 6). Possible evolutionary and biochemical An extensive body of previous work has endeavored to features of these pathways are discussed in more detail understand the phenomenon of SOS-induced mutagenesis elsewhere (14, 41). (iii) The inefficiency of the bypass reac- (1-4, 14). Mutagenesis requires a special set of proteins- tion may reflect the lack of another protein constituent that UmuC, UmuD', and RecA-that are not required for normal aids the pathway. Recent work has implicated the GroEL and DNA replication (6-12, 15-17). Because SOS mutagenesis is GroES heat shock proteins in SOS mutagenesis, with indi- associated with a response to a replication-inhibiting DNA cations for an interaction with UmuC (46, 47). These proteins lesion, the activity of the ancillary mutagenesis proteins has might be involved in the assembly and/or disassembly of the been postulated to involve replicative bypass of the DNA mutasome; alternatively, or in addition, heat shock proteins lesion, perhaps by an altered, relaxed-fidelity form of DNA might be required for efficient folding of a critical component polymerase (14, 40, 41). We have suggested (14, 17) that this such as UmuC. We have recently developed an in vitro multiprotein pathway might function through a damage- folding reaction for UmuC that requires the sequential action localized nucleoprotein complex-a "mutasome" (Fig. 6). of the DnaJ, DnaK, and GrpE heat shock proteins, followed Biochemistry: Rajagopalan et aL Proc. Natd. Acad. Sci. USA 89 (1992) 10781 by GroEL and GroES (M.-A. Petit, C.L., and H.E., unpub- 18. Bridges, B. A. & Woodgate, R. (1985) Proc. Nat!. Acad. Sci. lished work). USA 82, 4193-4197. Other in vitro work with randomly located abasic lesions 19. Lu, C. & Echols, H. (1987) J. Mol. Biol. 196, 497-504. has indicated a more efficient bypass without the mutagenesis 20. Bridges, B. A., Mottershead, R. P. & Sedgwick, S. G. (1976) proteins than we have observed (48); we presume that the Mol. Gen. Genet. 144, 53-58. 21. Hagensee, M. E., Timme, T., Bryan, S. K. & Moses, R. E. difference reflects the vastly different experimental condi- (1987) Proc. Natl. Acad. Sci. USA 84, 4195-4199. tions. Experiments in vivo using M13 ssDNA with a single 22. Witkin, E. M. (1970) Nature New Biol. (London) 229, 81-82. abasic site have revealed a 5% replicative bypass under 23. Bates, H., Randall, S. K., Rayssiguier, C., Bridges, B. A., SOS-induced conditions (43). Thus, the intracellular bypass Goodman, M. F. & Radman, M. (1989) J. Bacteriol. 171, reaction does remain highly restricted even when aided by 2480-2484. the complete SOS pathway. 24. Bonner, C. A., Randall, S. K., Rayssiguier, C., Radman, M., An interesting feature of the biochemical reaction is the Eritja, R., Kaplan, B. E., McEntee, K. & Goodman, M. F. apparent loss of processivity by pol III holoenzyme after (1988) J. Biol. Chem. 263, 18946-18952. bypassing the abasic lesion. Release of a relaxed-fidelity 25. Bonner, C. A., Hays, S., McEntee, K. & Goodman, M. F. polymerase makes sense in biological terms; a mutagenic (1990) Proc. Natl. Acad. Sci. USA 87, 7663-7667. DNA polymerase would constitute a liability during normal 26. Iwasaki, H., Nakata, A., Walker, G. C. & Shinagawa, H. DNA replication. However, we cannot yet say whether this (1990) J. Bacteriol. 172, 6268-6273. the limi- 27. Randall, S. K., Eritja, R., Kaplan, B. E., Petruska, J. & observation pertains to biological regulation or to Goodman, M. F. (1987) J. Biol. Chem. 262, 6864-6870. tations of our current biochemical assay. 28. Maki, H. & Kornberg, A. (1985) J. Biol. Chem. 260, 12987- Clearly, much remains to be learned about replicative 12992. bypass ofDNA lesions and its mutagenic consequences. The 29. Studwell, P. S. & O'Donnell, M. (1990) J. Biol. Chem. 265, availability of a biochemical reaction with pure proteins 1171-1178. should substantially accelerate progress. 30. Maki, S. & Kornberg, A. (1988)J. Biol. Chem. 263, 6555-6560. 31. Maki, H., Maki, S. & Kornberg, A. (1988) J. Biol. Chem. 263, We thank Richard Eisner for editorial help, Robert Lehman and 6570-6578. 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