Proc. Natl. Acad. Sci. USAVol. 86, pp. 7301-7305, October 1989Biochemistry

UmuC mutagenesis protein of Escherichia coli: Purification andinteraction with UmuD and UmuD'

(SOS response/fidelity of DNA replication/protein-protein interaction)

ROGER WOODGATE, MALINI RAJAGOPALAN, CHI Lu, AND HARRISON ECHOLSDepartment of Molecular Biology, University of California, Berkeley, CA 94720

Communicated by Evelyn M. Witkin, May 30, 1989

ABSTRACT The introduction of a replication-inhibitinglesion into the DNA of Escherichia coli produces a markedelevation in mutation rate. The mutation pathway is a compo-nent of the induced, multigene SOS response. SOS mutagenesisis a tightly regulated process dependent on two RecA-mediatedproteolytic events: cleavage of the LexA repressor to induce theUmuC and UmuD mutagenesis proteins, and cleavage ofUmuDto UmuD' to activate the mutation pathway. To investigate theprotein-protein interactions responsible for SOS mutagenesis,we have studied the interaction of UmuC, UmuD, and UmuD'.To probe intracellular interaction, we have used immuno-precipitation techniques with antibodies against UmuC orUmuD and UmuD'. We have found that antibody to UmuCprecipitates UmuD' from cell extracts, and antibody to UmuDand UmuD' precipitates UmuC. Thus we conclude that UmuCprobably associates tightly with UmuD' in cells. For biochem-ical studies, we have purified the UmuC and UmuD' proteinsto use with the previously purified UmuD. UmuC associatesstrongly with an affinity column ofUmuD and UmuD', elutingonly under strongly dissociating conditions (2M urea or 1.5MKSCN). UmuC also associates efficiently with UmuD or UmuD'in solution, as judged by velocity sedimentation in a glycerolgradient. The likely stoichiometry is one UmuC with a dimericUmuD or UmuD'. From these experiments and previous work,we infer that SOS mutagenesis depends on the action of theUmuC-UmuD' complex and probably RecA to rescue a stalledDNA polymerase HI holoenzyme at the DNA lesion.

Mutagenesis of Escherichia coli by UV light and mostchemical agents occurs as part of the multigene inducibleSOS response to DNA damage (1-4). To initiate the SOSresponse, RecA protein is activated by DNA damage tomediate the proteolytic cleavage of the LexA repressor forSOS-controlled genes (2, 4). Genetic and physiological ex-periments indicate that the products of the umuDC operonand the recA gene are directly required for the mutationpathway (5-11). The individual roles of these proteins areunclear. The available evidence has indicated that there is afunctional and possibly physical interaction among UmuD,UmuC, RecA, and DNA polymerase III holoenzyme at thereplication-blocking lesion; this multiprotein pathway pre-sumably serves to effect translesion DNA replication, withthe introduction of mutations targeted to the damaged site(12-19).

Recently the framework of possible protein-protein inter-actions has been extended by the discovery that UmuDundergoes RecA-mediated cleavage to a product, UmuD',which is the active agent for SOS mutagenesis. We havepurified the UmuD protein and demonstrated the cleavagereaction in vitro (20). Other work has shown that RecA-dependent cleavage of UmuD occurs in vivo (21). Moreover,

an engineered umuD gene producing only UmuD' can restoreUV mutagenesis to cells with a mutant RecA protein unableto cleave UmuD (11).

Genetic evidence has indicated that UmuC and UmuD(and/or UmuD') interact at least functionally (16). We haveinvestigated the physical interaction by immunoprecipitationstudies in cell extracts and by association reactions withpurified proteins. For biochemical experiments, we havepurified UmuC and UmuD' proteins to use with the previ-ously purified UmuD. Our data indicate that UmuC andUmuD' associate strongly in vivo and in vitro. We haveconcluded that the UmuC-UmuD' complex is the activeagent for SOS mutagenesis.

MATERIALS AND METHODSMaterials. Staphylococcal protein A-Sepharose, mitomy-

cin C, tetracycline, and ampicillin were purchased fromSigma. Phosphocellulose was obtained from Whatman,DEAE-Sephacel from Pharmacia, hydroxylapatite and Affi-Gel 15 from Bio-Rad, AcA 54 from IBF, 3-[(3-cholamidopro-pyl)dimethylammonio]-1-propanesulfonate (CHAPS) fromPierce, and Gammabind G agarose from Genex. E. coli 30Sribosomal protein S9 was a kind gift of M. Nomura (Univer-sity of California, Irvine).Immunoprecipitation of Umu Proteins. Antisera to highly

purified UmuC and UmuD/UmuD' proteins were raised inrabbits by standard procedures. To produce UmuD, UmuD',and UmuC proteins, we used E. coli strain MC1000 contain-ing plasmids pRK248(AcIts) and pSB13 (overproducingUmuD and UmuC proteins) (20). Cells were grown in M9low-sulfur medium at 30'C to an OD6w of 0.2 (20). Mitomy-cin C was then added to a final concentration of 1 tkg/ml, andcultures were grown to an OD6w of 0.4. At this point,Na235SO4 was added at 100 ,uCi/ml (1 Ci = 37 GBq), and thetemperature was raised to 420C for 40 min to induce theUmuD- and UmuC-overproducing vector. The 35S-labeledcells (5 ml) were added to 1 ml of nonradioactive carrier cells(MC1000 with pRK248 and pRC23) (22), which had previ-ously been grown overnight in LB broth, concentrated 10-fold in M9 low-sulfur medium, treated with 3 mM N-ethylmaleimide and phenylmethylsulfonyl fluoride (PMSF)at 200 pug/ml for 10 min at 370C, and supplemented with 10mM NaN3. The cells were then harvested, frozen, andthawed in 50 1.I of buffer containing 10 mM Tris HCI at pH8.0, 0.1 mM dithiothreitol, 50 mM KCI, and lysozyme at 0.5mg/ml. The cells were lysed by freezing and thawing fivetimes. Further preparation of lysates was as previouslydescribed (23). To lower nonspecific adsorption, the extractswere incubated with cII antisera and protein A-agarose for 1hr at room temperature and then centrifuged. The pellet wasdiscarded, and the supernatant fraction was transferred to afresh tube and incubated for 4 hr on ice with an excess of cII,

Abbreviation: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.

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7302 Biochemistry: Woodgate et al.

UmuC, or UmuD antiserum and 50 td of Gammabind Gagarose in 150 ,ul of PBS/azide (10 mM sodium phosphate,pH 7.4/150 mM NaCl/0.05% NaN3). Immunoprecipitateswere collected by centrifugation and washed five times withPBS/azide. The immunoprecipitated proteins were removedfrom the final pellet by boiling 5 min in 60 gI of 2 x Laemmlisample buffer (24). Proteins were separated by electropho-resis in NaDodSO4/9-19% polyacrylamide and analyzed byautoradiography.

Preparation of UmuD/UmuD' Protein Affinity Column.Strain MC1000 (pRK248, pSB13) was grown, induced, har-vested, and lysed as described (20), except that mitomycin Cwas added to a final concentration of 1 pug/ml at a cell densityof 2 x 107 per ml. The purification was essentially thatdescribed previously for UmuD (20), except that the order ofcolumn steps was DEAE-Sephacel, hydroxylapatite, AcA54. The purified UmuD and UmuD' proteins were cross-linked to Affi-Gel 15 affinity support beads according to theprocedure of Formosa et al. (25).

Purification ofUmuD' Protein. UmuD' protein was purifiedby a procedure similar to that used for the UmuD/UmuD'mix. Cells were treated with mitomycin C at 5 gg/ml prior totemperature induction of the overproducing system to ensurefull activation of RecA and maximize UmuD cleavage. Lysisand purification up to and including the wash of the hydrox-ylapatite column were the same as for the purification of theUmuD/UmuD' mixture. After the hydroxylapatite columnwas washed with phosphate buffer, the column was devel-oped with 8 column volumes of a linear 20-200 mM NaHPO4gradient, pH 6.8. Additional UmuD' free of UmuD eluted at40 mM NaHPO4. UmuD' fractions were pooled, concen-trated by precipitation with ammonium sulfate, and applied toa 100-ml (1.4 cm x 65 cm) AcA 54 gel filtration column aspreviously described (20).

Purification of UmuC Protein. A 100-liter culture of strainMC1000 (pRK248, pSB13) was grown, induced, and lysed aspreviously described (20). The lysed cells were centrifuged at240,000 x g for 2 hr, after which time greater than 95% of theinduced UmuC present in the harvested cells remainedassociated with the cell debris (as judged by staining withCoomassie blue). A portion (5 g) of the cell debris washomogenized with a blender after the additional of 50 ml ofR buffer [20 mM Tris HCl, pH 7.5/50 mM NaCI/0.1 mMEDTA/5 mM dithiothreitol/10% (vol/vol) glycerol] contain-ing 1 M NaCl. After centrifugation at 140,000 x g for 30 min,the pellet was resuspended, homogenized, and recentrifugedtwice more with R buffer containing 1 M NaCI and then oncewith 50 ml ofR buffer. UmuC was converted to a soluble formby denaturation with 8 M urea; 1 g of the cell pellet washomogenized in 10 ml of P buffer (20 mM KHPO4, pH 6.5/1mM dithiothreitol/8 M urea/10% glycerol) and centrifuged at140,000 x g for 30 min. The resulting supernatant was applieddirectly to a 10-ml phosphocellulose column equilibrated in Pbuffer. The column was washed with 6 column volumes of Pbuffer and then with 6 column volumes ofP buffer containing500 mM KCL. UmuC eluted in P buffer containing 500 mMKCL. The UmuC protein was then renatured. UmuC-containing fractions were pooled, and the UmuC sample wasdiluted 1:4 by adding it slowly with stirring to R buffercontaining 1 M NaCl and 12 mM CHAPS, at 20°C, followedby stirring for an additional 15 min. During the dilution stepa precipitate formed. The solution was cleared by centrifu-gation (140,000 x g for 30 min), and the supernatant fractionwas dialyzed against C buffer (20 mM Tris HCl, pH 7.5/200mM NaCl/0.1 mM EDTA/1 mM CHAPS/10%o glycerol/5mM dithiothreitol). About 90% of the UmuC was lost in therenaturation procedure. The dialyzed sample was applied toa 1-ml protein affinity column containing UmuD and UmuD'(3.6 mg of protein) at a flow rate of 4 ml per hour. The columnwas washed with 10 column volumes ofC buffer, followed by

10 column volumes of the same buffer containing 2 M NaCl.The column was then reequilibrated in C buffer, followed byelution of UmuC with C buffer containing 2 M urea or 1.5 MKSCN. UmuC-containing fractions were pooled and diluted1:5 in R buffer containing 1 M NaCl and 12 mM CHAPS andan equimolar amount of ribosomal protein S9. The resultantsolution was dialyzed against R buffer containing 0.2 mMCHAPS, during which time a precipitate formed (consistingmainly of S9 protein). The solution was cleared by centrifu-gation in a micro-centrifuge at 14,000 x g. The supernatantwas removed and concentrated by reverse dialysis.

Glycerol Gradient Analysis of Protein Complexes. UmuCprotein (4 ug) was incubated with UmuD or UmuD' (10 Ag)at 370C for 5 min. Samples were then applied to a 10-30%(vol/vol) linear glycerol gradient in R buffer (4 ml). Proteinswere separated by centrifugation in a Beckman SW56 rotorat 49,000 rpm at 40C for 21 hr. Fractions (110 ,Al) werecollected, and all of the fraction was applied to a NaDodSO4/9-19% polyacrylamide gel. Proteins were visualized by stain-ing with Coomassie brilliant blue R250.N-Terminal Amino Acid Analysis of Proteins. Proteins were

separated on a NaDodSO4/9-19% polyacrylamide gel andwere transferred to polyvinylidenedifluoride membranes asdescribed (26). Amino acid sequence analysis was kindlyperformed by Sigurd Wilbanks and Arie Admon at theUniversity of California, Berkeley.

RESULTSAssociation of UmuC with UmuD' in Vivo. To investigate the

possible intracellular interaction of UmuC, UmuD, andUmuD', we have used immunoprecipitation techniques. Thetest for protein-protein association was the capacity ofantibody to UmuD and UmuD' to precipitate UmuC from cellextracts, and the reciprocal ability of antibody to UmuC toprecipitate UmuD and UmuD'. The antibody to UmuD/UmuD' does not cross-react with free UmuC, nor doesUmuC antibody cross-react with UmuD or UmuD'; thus,coprecipitation of the proteins from 35S-labeled extracts wastaken to indicate that the proteins were likely to be associatedin the intracellular environment.For the immunoprecipitation experiments, cells were ther-

mally induced for overproduction of UmuC and UmuD aftertreatment with mitomycin C to produce cleavage of UmuD(under conditions in which UmuD and UmuD' were pro-duced in approximately equal quantities). Treatment withantibody to A cII protein failed to precipitate any of the Umuproteins (Fig. 1, lane 1). Antibody to UmuC coprecipitatedUmuD' (Fig. 1, lanes 2 and 4); coprecipitation of UmuD wasnot apparent (there is a faint, presumably contaminating,band also present in the cII lane). Antibody to UmuD andUmuD' coprecipitated UmuC (Fig. 1, lane 3). Because nei-ther UmuC nor UmuD' was coprecipitated by the antibody toA cII protein, we take the coprecipitation as evidence of aspecific interaction. There is much less UmuC in lane 2 thanUmuD and UmuD' in lane 3 because most of the overpro-duced UmuC is insoluble (as noted in the purification ofUmuC). From scanning the gels of lanes 2 and 4, we estimatea molar ratio of about 2.5 UmuD' to 1 UmuC, based oncysteine and methionine content of the two proteins (UmuChas 23 ofthese amino acids to 3 for UmuD'). Thus, the solubleUmuC appears to be tightly associated with UmuD'. As aconsequence of the deficit in soluble UmuC, the coprecipi-tation assay with UmuC antibody would not pick up aUmuC-UmuD interaction if it were even slightly disfavoredwith respect to UmuC-UmuD'. In another experiment withextracts in which UmuD was present in excess to UmuD',only coprecipitation of UmuD' by UmuC antibody wasobserved (data not shown). From the results of Fig. 1, we

Proc. Natl. Acad. Sci. USA 86 (1989)

Proc. Natl. Acad. Sci. USA 86 (1989) 7303

Precipitation by Antibody toClH UmuC UmuD UmuC

-I o_ FUmuCAWW

.4.. ...

- UmuD

(- UmuD'

FIG. 1. Immunoprecipitation of proteins from cell extracts byusing antibodies against UmuC, UmuD, and A clI proteins. Proteinswere fractionated by electrophoresis on a NaDodSO4/9-19% poly-acrylamide gel and visualized by autoradiography. Lanes: cHI anti-body; UmuC antibody; UmuD and UmuD' antibody; UmuC anti-body with additional background clearing step. The positions ofUmuD, UmuD', and UmuC are marked on the right.

conclude that there is likely to be an intracellular interactionbetween UmuC and UmuD'.

Purification of UmuD' and Determination of Cleavage Site.To construct an affinity column with UmuD and UmuD', wepurified a mixture of the two proteins by a slight modificationof the procedure used previously for UmuD (20). MitomycinC was used at relatively low concentration (1 jug/ml) toprovide a moderately active RecA-mediated cleavage reac-tion, resulting in approximately equal quantities of UmuDand UmuD'. To purify UmuD', we increased the concentra-tion of mitomycin C (5 ,ug/ml) to provide for a higherconcentration of the cleaved protein. Purified UmuD' wasseparated from a fraction with UmuD and UmuD' by the finalstep of hydroxylapatite chromatography. The N-terminalamino acid sequence ofUmuD' was determined to define thecleavage site in the proteolytic reaction. The sequence of thefirst nine amino acids was Gly-Phe-Pro-Ser-Pro-Ala-Ala-Asp-Tyr. The complete amino acid sequence of UmuDhas been deduced from the DNA sequence (16, 27). Weconfirmed the first nine amino acids of the UmuD sequence.The sequence of UmuD' established the cleavage point as aCys-Gly bond at amino acid 24 from the N terminus; this sitewas predicted from a comparison of the amino acid sequenceof UmuD with that found near the cleavage site of LexA andA cI (16). These data support previous conclusions that theRecA-mediated proteolytic reactions proceed by closely sim-ilar reaction pathways, involving a facilitated self-cleavagemechanism (20, 28, 29).

Purification of UmuC. The UmuC was purified from cellscarrying an overproducing plasmid system (20). The purifi-cation was followed by polyacrylamide gel electrophoresis inNaDodSO4. Nearly all of the UmuC was found in themembrane fraction of the lysed cells. This observation is acommon finding for overproduced proteins (e.g., see ref. 30and references therein). This membrane localization provedadvantageous in that a high degree of purification wasachieved by washing the cell pellet with 1 M NaCl; UmuC andUmuD were the main remaining proteins. Solubilization ofUmuC from the membrane fraction was achieved only bydenaturing conditions of 8M urea. UmuC was separated fromUmuD in the denatured state by adsorption on a phospho-cellulose column in 8M urea, followed by elution in the samebuffer containing 500mM KCl. UmuC was then renatured bydilution and subsequent dialysis.

The final step in the purification of UmuC was affinitychromatography with a column of UmuD/UmuD' (Fig. 2).Most proteins in the renatured fraction (Fig. 2, lane L) werenot retained on the column (Fig. 2, lane F). Elution of thecolumn with buffer containing 2 M NaCI removed severalproteins and a small amount of UmuC (Fig. 2, lane S). Mostof the UmuC remained bound to the column and could beeluted only by using buffer containing strongly dissociatingreagents, 2 M urea or 1.5 M KSCN (Fig. 2, lane U). Toconfirm the identity of UmuC, the N terminus of the purifiedprotein was sequenced (9 amino acids) and shown to beidentical to that predicted by DNA sequence data (16, 27). Tobe sure that the interaction observed with the affinity columnwas specific, we carried out an equivalent experiment with anaffinity column containing bovine serum albumin instead ofUmuD/UmuD'. Bovine serum albumin has approximatelythe same isoelectric point as UmuD/UmuD', thus controllingfor nonspecific charge effects. We found that UmuC waseluted in the flow-through fraction (data not shown). Fromthe affinity column results, we conclude that UmuC associ-ates tightly with UmuD and/or UmuD'.Although the UmuC applied to the affinity column was

soluble, the UmuC that was eluted from the affinity columnhad low solubility, precipitating soon after elution. We be-lieve that our purification protocol might have removed aprotein that stabilizes UmuC in solution. Candidates werethose proteins that were retained on the column but wereeluted with 2 M NaCl (Fig. 2, lane S). We determined theN-terminal sequence of the most abundant of the proteins inthe 2 M NaCl fraction. The sequence matched that of 30Sribosomal protein S9. To test the hypothesis that S9 stabilizesUmuC protein, we obtained highly purified S9 from M.Nomura. We found that addition of S9 protein to the affinitycolumn eluate prevented precipitation of the UmuC protein,although addition of a number of other proteins (includingUmuD) did not. UmuC and S9 were separated by sedimen-tation in a glycerol gradient, indicating that either a weakphysical interaction or some transient activity of S9 proteinwas involved in stabilizing UmuC in solution (data notshown).

Association of Purified UmuC with UmuD and UmuD'. Tostudy association of the purified UmuC with UmuD and

L F S U

68 -

45- _ E_ UmuC

25 _

1 4

.....s(- S9

FIG. 2. Affinity chromatography of UmuC. Proteins were frac-tionated by electrophoresis on a NaDodSO4/9-19%o polyacrylamidegel and visualized by Coomassie blue. Lanes: L, the fraction appliedto the UmuD/UmuD' affinity column (16 ,tg was loaded); F, flow-through fraction (12 ,ug); S, fraction eluted with 2 M NaCl (16 pg);U, fraction eluted with 2 M urea (6 ,ug) (similar results were obtainedwith 1.5 M KSCN). The positions of UmuC and S9 proteins aremarked on the right; the positions and apparent molecular masses (inkDa) of marker proteins are given on the left.

Biochemistry: Woodgate et al.

7304 Biochemistry: Woodgate et al.

UmuD', we used velocity sedimentation in a glycerol gradi-ent. The molecular mass of UmuC predicted from the DNAsequence is 48 kDa (16, 27). Purified UmuC alone sedimentedat a velocity expected for a protein ofabout 45 kDa, asjudgedby the sedimentation of marker proteins of known molecularmass (Fig. 3, upper gel). Thus UmuC appears to be amonomer in solution. After mixing with a molar excess ofUmuD', UmuC sedimented at nearly the same velocity as amarker ofbovine serum albumin (68 kDa), consistent with theformation of a UmuC-UmuD' complex with a molecularmass of about 70 kDa (Fig. 3, lower gel). The fraction ofUmuD' migrating with bovine serum albumin is also consis-tent with a complex of UmuC and UmuD', because this morerapidly sedimenting fraction was not seen in a gradient withonly UmuD' (data not shown). As judged by sedimentationand gel filtration studies, UmuD' (12 kDa) alone is probablyan asymmetric dimer; UmuD' sediments more slowly than aglobular dimer, but it migrates in gel filtration as somewhatlarger than a globular dimer (our unpublished data). Thus theUmuC-UmuD' complex is likely to contain one UmuC andtwo UmuD' polypeptides. However, further work will berequired to establish a rigorous stoichiometry. After mixingwith a molar excess of UmuD, UmuC sedimented slightlymore rapidly than bovine serum albumin, consistent withformation of a UmuC-UmuD complex (data not shown).From the affinity column and sedimentation data, we

conclude that UmuC in solution associates tightly with eitherUmuD or UmuD'.

DISCUSSIONProtein-Protein Interactions of UmuC, UmuD, and UmuD'.

We have shown that UmuC is found associated with UmuD'in cell extracts and that purified UmuC forms a complex withUmuD' in solution. Because UmuD must be cleaved toUmuD' to turn on the mutagenic pathway, we conclude thatthe UmuC-UmuD' complex is probably the active agent forSOS mutagenesis. UmuC associates also with UmuD insolution. We did not find evidence for a UmuC-UmuD

12 kDa 45 kDa 68 kDa

*BSA

..UmuC

-BSA

<- UmuC

*_UmuD'

FIG. 3. Glycerol gradient analysis of UmuC (upper gel) and amixture of UmuD' and UmuC (lower gel). Fractions were collected,and proteins were fractionated by electrophoresis on NaDodSO4/9-19% polyacrylamide gels and visualized by Coomassie blue. Thepositions ofUmuC and UmuD' are marked on the right, together withthe position of bovine serum albumin (BSA), which was included inthe gradients as an internal reference marker; the positions andapparent molecular masses (in kDa) of other marker proteins from aseparate gradient are given at the top of the figure. Only the top (mostslowly sedimenting) 18 fractions of a total of 37 fractions are shown.

complex in cell extracts. This observation might mean thatthe UmuC-UmuD' complex is more stable or more solublein vivo or has other properties favored by the immunoprecip-itation reaction.One intriguing and puzzling interaction is that involving

UmuC and ribosomal protein S9. The most interesting pos-sibility is that S9 is somehow involved in assembling themultiprotein complex required for translesion DNA replica-tion. S9 would be retained on the affinity column because ofa weak specific association with UmuC (and/or UmuD/UmuD'). This postulated role would be analogous to the needfor ribosomal protein S10 in the assembly of the antitermi-nation complex for transcription of phage A (31). Anotherinteresting but less specific role for S9 would be a "renatu-rase" function. UmuC might be partially unfolded by thestringent elution conditions from the affinity column (orincompletely refolded after renaturation); S9 might thensupply a weakly interacting hydrophobic domain to allowUmuC to refold before self-associating by exposed hydro-phobic regions and precipitating. In the absence of a bio-chemical assay for UmuC, we cannot be sure that UmuC hasbeen completely renatured. Two observations are consistentwith a native conformation: (i) UmuC sediments as a globularmonomer of the expected molecular mass, (ii) UmuC asso-ciates with UmuD' to give a complex sedimenting as ex-pected for a stable and specific interaction.

Possible Mechanisms for SOS Mutagenesis and RelatedRescue Functions. Although our experiments indicatestrongly that the UmuC-UmuD' complex is required for themutation pathway, the mechanism for SOS mutagenesisremains undefined. However, taken together with otherrecent work, our new information about UmuC-UmuD'allows us to suggest a rather explicit model that is subject tobiochemical analysis with currently available proteins andDNA templates (Fig. 4). This proposed mechanism is arefinement of previous models (3, 32).DNA polymerase III holoenzyme stops replicating DNA at

most bulky-adduct lesions (e.g., dipyrimidine UV lesions) or

TIOUl | 0

UrnuC/Dj RecA

A

Cleave UrmuD IL1?Z f7S7i;zCleave LexA(mutagenesis) (induce SOS)

UmuC/D I Pol III

XTC~ ~ ~ ~ ~ _T

Translesion replication

Replication restart(strand switch?)

FIG. 4. Possible mechanism of SOS-induced mutagenesis. Amultiprotein complex is assembled at the DNA lesion, includingUmuC, UmuD', RecA, and DNA polymerase III holoenzyme (PolIII). This nucleoprotein aggregate provides for replication across thelesion with the introduction of mutations. A possible alternativepathway for error-free replicative bypass is DNA synthesis with thedaughter strand as template in a three-stranded complex generatedby RecA.

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Proc. Natl. Acad. Sci. USA 86 (1989) 7305

apurinic sites (33-35). The polymerase presumably stalls fortwo reasons: the distorted template does not allow effectivebase-pair recognition for base insertion by the polymerase;and even a correctly inserted base looks like a mismatch tothe exonucleolytic editing subunit. The mutation pathwaypresumably involves successful replication across this dis-torted site with the introduction of errors (translesion repli-cation) (3, 36).SOS mutagenesis requires the activity of UmuC-UmuD'

and probably a "direct role" of RecA (an activity in additionto cleavage of LexA and UmuD) (refs. 16 and 37; E. M.Witkin, personal communication). We presume that replica-tive bypass depends on formation of a lesion-localized nu-cleoprotein structure involving RecA, UmuC-UmuD', andDNA polymerase III (Pol III) (Fig. 4). This multiproteincomplex might facilitate bypass replication in several ways:(i) a change in the template conformation to compensate forthe distortion and allow base insertion by Pol III; (ii) aninhibition of the editing exonuclease; (iii) facilitated bindingby Pol III at the distorted primer-template region, preventingdissociation and permitting multiple rapid replication at-tempts; (iv) an alteration in the enzyme to relax the require-ments for precise base pairing (3, 14, 17, 32, 35, 36, 38). Thefirst three possibilities seem relatively likely, and all fourmight be involved.A possible pathway for assembly of the "mutasome"

structure is presented in Fig. 4. RecA filaments form at thesite of the lesion because RecA recognizes the lesion-induceddistortion in the DNA duplex (17, 32). RecA is activated byassociation with DNA to cleave LexA and induce synthesisof UmuC and UmuD; UmuD is then cleaved to UmuD'. Atthe site of the DNA lesion, a multiprotein complex is assem-bled, including UmuC, UmuD', RecA, and DNA polymeraseIII holoenzyme. An alternative assembly pathway for thesame nucleoprotein complex involves RecA binding to sin-gle-strand DNA generated downstream from the lesion afterthe polymerase stalls (39) (bottom of Fig. 4). UmuC is a highlybasic protein, and so its primary function may be to bringUmuD to the DNA for cleavage and/or to help maintainUmuD' at the lesion site (our preliminary experiments indi-cate that UmuC does bind to DNA).The nucleoprotein assembly depicted at the bottom of Fig.

4 might also serve to provide for the "replication-restart"function of RecA protein, the capacity to resume replicationafter DNA damage by an error-free pathway (40, 41). TheRecA-coated single-strand DNA downstream from theblocked polymerase will likely associate with the replicatedduplex from the complementary strand. In the resultantthree-stranded structure, the polymerase could bypass thelesion by copying the undamaged daughter strand instead ofthe blocked parental strand, switching back after clearing thelesion (3, 32) (if replication on the daughter strand proceededfar enough to interwind the two strands, a breaking andjoining event would also be required). The proposed repli-cation-restart pathway would augment and might sometimesinitiate the previously defined recombinational repair path-way (42). As judged by UV sensitivity of mutants, thereplication-restart pathway is much more important for cel-lular survival than the mutational pathway (41). The muta-tional pathway might serve primarily as a population rescuefunction, by introducing enhanced genetic variation (e.g., seeref. 3).

We thank Masayasu Nomura for E. coli ribosomal protein S9; ArieAdmon and Sigurd Wilbanks for amino acid sequencing; HiroshiNikaido, Edwin Eisenstein, Dave Sloane, and Dan Milligan forhelpful suggestions on protein purification; Marcie Rosenberg forcomments on the manuscript; Chris Vasilikiotis, Mimi Meyers, and

Ellis Meyers for photography; and Richard Eisner for editorial help.This work was supported in part by grants from the National CancerInstitute (CA 41655), the National Institute of Environmental HealthSciences (ES 01896), the University of California Cancer ResearchCoordinating Committee; by a Wellcome Trust Travel Grant toR.W.; and by a senior postdoctoral fellowship to R.W. from theAmerican Cancer Society, California Division.

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