Proc. Natl. Acad. Sci. USAVol. 85, pp. 1816-1820, March 1988Biochemistry

RecA-mediated cleavage activates UmuD for mutagenesis:Mechanistic relationship between transcriptionalderepression and posttranslational activationTAKEHIKO NOHMI, JOHN R. BATTISTA, LORI A. DODSON, AND GRAHAM C. WALKERBiology Department, Massachusetts Institute of Technology, Cambridge, MA 02139

Communicated by Evelyn M. Witkin, November 30, 1987

ABSTRACT The products of the SOS-regulated umuDCoperon are required for most UV and chemical mutagenesis inEscherichia coli. It has been shown that the UmuD proteinshares homology with LexA, the repressor of the SOS genes.In this paper we describe a series of genetic experiments thatindicate that the purpose of RecA-mediated cleavage ofUmuDat its bond between Cys-24 and Gly-25 is to activate UmuD forits role in mutagenesis and that the COOH-terminal fragmentof UmuD is necessary and sufficient for the role of UmuD inUV mutagenesis. Other genetic experiments are presented that(i) support the hypothesis that the primary role of Ser-60 inUmuD function is to act as a nucleophile in the RecA-mediatedcleavage reaction and (ii) raise the possibility that RecA has athird role in UV mutagenesis besides mediating the cleavage ofLexA and UmuD.

Most mutagenesis of Escherichia coli by UV irradiation anda variety of chemicals requires the UmuD and UmuCproteins (1-4) or their plasmid-derived analogs MucA andMucB. Both umuD and umuC mutants are virtually nonmu-table with UV and many chemicals (1, 5, 6). The umuD andumuC genes are organized in an operon (1, 2) and encodeproteins of 15.0 and 47.7 kDa, respectively (7, 8). The mucAand mucB genes of the plasmid pKM101 are also organizedin an operon and encode similarly sized products (7, 9).Both the umuDC operon and the plasmid-derived mucAB

operon are repressed by the LexA protein (1, 2, 10) andregulated as part of the SOS response of E. coli (3, 4, 11, 12).SOS induction occurs by activated RecA (designatedRecA*) mediating the proteolytic cleavage of LexA at itsbond between Ala-84 and Gly-85 (13) apparently by facilitat-ing a specific autodigestion of LexA (14). Slilaty and Little(15) have recently suggested that hydrolysis of the LexAAla-Gly bond proceeds by a mechanism similar to that ofserine proteases, with Ser-119 acting as a nucleophile andLys-156 as an activator. LexA shares homology with therepressors of bacteriophages A, 434, P22, and 480 (16, 17),and cleavage of these proteins appears to occur by ananalogous mechanism (17-19). The cleavage site of all theseproteins is an Ala-Gly bond except for the 480 repressor,which has a Cys-Gly cleavage site (17).UmuD and MucA share homology with the COOH-

terminal regions ofLexA and these phage repressors (7). Thisled our laboratory to propose that UmuD and MucA mightinteract with RecA* and that this interaction could result in aproteolytic cleavage of these proteins that would activate orunmask their function required for mutagenesis (7). Theputative cleavage site of UmuD is the bond between Cys-24and Gly-25, while the putative cleavage site of MucA is thebond between Ala-25 and Gly-26. Furthermore, the verylimited homology between the amino acids of UmuD and

MucA on the NH2-terminal side of their putative cleavagesites led to the suggestion that they might constitute anonfunctional or expendable domain (7). Recent analyses of amucA101 (Glu-26) mutant have supported the above hypoth-esis (20).The possibility that RecA mediates the cleavage of UmuD

and MucA has taken on added significance in the face of agrowing body of evidence that RecA is required for at leastone other role in mutagenesis besides mediating the cleavageof LexA (3). This was first suggested by the observation thatdefective chromosomal lexA(Def) recA(Def) strains are non-mutable despite the lack of functional LexA (10, 12, 21). Aneed for RecA to be activated for an additional role besidescleaving LexA has been suggested by analyses of mutagenesisin strains carrying various recA mutations that alter the abilityof RecA to be activated (3, 22-25) and by various physiolog-ical experiments (26, 27). Recent work in this laboratory (20)suggests that RecA may play at least two additional roles inmutagenesis besides mediating the cleavage of LexA.The accompanying papers (28, 29) demonstrate that the

proposed RecA-mediated cleavage of UmuD does indeedoccur. In this paper, we present genetic evidence indicatingthat the purpose of this cleavage is to activate UmuD for itsrole in mutagenesis and suggesting that the COOH-terminalcleavage product is necessary and sufficient for this role.

MATERIALS AND METHODSPlasmid pDS101 was constructed by cloning the umuD',C+-containing Cla I-Cla I fragment of pSE117 (1) into theCla I site of pZ150 (30). Plasmid pGW2101 was constructedby cloning the umuD+ ,C'-containing Hpa I-Hpa I fragmentof pSE117 into the EcoRV-Pvu II fragment of pZ150.Plasmid pGW2020 was derived by deleting umuC frompGW2101 by BamHI digestion, partial Bgi II digestion, andreligation. Mutants of umuD were constructed by an oligo-nucleotide-directed mutagenesis system (Amersham) usingoligonucleotides ranging in length from 18 to 32 bases andwere confirmed in each case by sequencing of the entireumuD gene. GW3200 is AB1157 umuD44 (1). GW2735 is alexA7J: :Tn5 recA + (31) derivative of GW1000 (10). GW6900is a lexA7J::TnS recA430 derivative of GW1000 constructedby C. Donnelly (Massachusetts Institute of Technology).GW6752 is a lexA71::TnS A(recA-sriR)306::TnJO derivativeofGW1000 constructed from GW2735 by phage P1 transduc-tion from DE140 provided by D. Ennis (University ofArizona). UV mutagenesis was carried out as describedpreviously except that, in the case of the lexA71 :TnSA(recA-sriR)306::TnJO strains, cells were concentrated 10 or100 times before plating as indicated (1). The maxicelllabeling was carried out with strain CSR603 (32).

RESULTS

Homology of UmuD to LexA and Phage Repressors HasFunctional Significance. To test the significance of the ho-

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Proc. Natl. Acad. Sci. USA 85 (1988) 1817

Table 1. Summary of mutant plasmidsPlasmid* Mutationt Amino acid change

70 24pLD1009 TGT TAT Cys Tyr

73 25pGW2110 GGC - GAA Gly Glu

73 25pGW2111 GGC AAA Gly Lys

178 60pGW2112 TCT - GCT Ser-- Ala

178 60pGW2113 TCT TGT Ser-- Cys

298 97pGW2115 AAA GCA Lys-- Ala

70 24 25 25pGW2117 TGTGGC -- TGATGGGC Cys-Gly- stop(fMet)-Gly

70 24 25pGW2119 TGTGGC - TGATGGC Cys-Gly- stop-frameshift

25pGW2122 A nucleotides 4-72 Wild type -. (fMet)-Gly

70 24 25 25pGW2118 TGTGGC -- TGATGGGC Cys-Gly--+ stop(fMet)-gly

178 60TCT -* GCT Ser- Ala

70 24 25 25pGW2121 TGTGGC TGATGGGC Cys-Gly-* stop(fMet)-Gly

298 97AAA GCA Lys-- Ala

*pLD1009 was obtained following treatment of pDS101 with hy-droxylamine. Plasmids pGW2110-pGW2115 were generated byoligonucleotide-directed mutagenesis of pGW2101. PlasmidspGW2117-pGW2122 were generated by oligonucleotide-directedmutagenesis of pGW2020.tNumbering of umuD is that of Perry et al. (7).

mology of UmuD to LexA and certain phage repressors (7),we used site-directed mutagenesis of a umuD+,C' plasmidto create certain umuD mutations that were analogous tolexA or A repressor mutations that block both RecA-mediated cleavage and autodigestion (Table 1 and Fig. lA).We then examined the ability of the plasmids carrying theseumuD mutations to complement a UV-nonmutable umuD44strain (Fig. 2A). We found that changing the Gly-25 residueof the putative Cys-Gly UmuD cleavage site to a glutamic orlysine residue largely abolished the ability of UmuD tofunction in UV mutagenesis. A Gly -+ Glu change at theAla-Gly cleavage site of A repressor completely blockscleavage (33), while a corresponding Gly -+ Asp change inLexA largely blocks cleavage (13, 14). A mutation, isolatedafter hydroxylamine mutagenesis, that changed Cys-24 to atyrosine residue caused a less severe reduction of the abilityof UmuD to function in UV mutagenesis. In addition, we

A H2N- gIn23cys24gly25 ser60 Iys97

tyr glu lys ala cys ala

B H2N gln23COOHH2N(f-met)gly25

C H2N gln23COOH

D H2NIT-met)gly25

E H2N gln23COOHH2N(f-met)gly25 ser60 Iys97

ala ala

found that changing either Ser-60 or Lys-97 to an alanineresidue also greatly reduced UmuD's ability to function inUV mutagenesis. Slilaty and Little (15) have shown thatchanges of the corresponding Ser-119 and Lys-156 residuesof LexA to alanine residues completely block cleavage.They also reported that changing Ser-119 to a cysteineresidue, another residue with a nucleophilic sidechain, didnot block LexA cleavage as severely as the Ser-119 -+ Alachange. Interestingly, we found in UmuD that the Ser-60 --

Cys change caused a less severe reduction in the ability ofUmuD to function in mutagenesis than the Ser-60 -_ Alachange (Fig. 2A).Thus, all of these umuD mutations caused major reduc-

tions in the ability of UmuD to function in UV mutagenesis.Since by analogy to LexA and phage A repressor, theseumuD mutations might be expected to block or reduceRecA-mediated cleavage, these results suggest that cleavageof UmuD at the bond between Cys-24 and Gly-25 is impor-tant for UV mutagenesis. The more severe effect of theSer-60 -- Ala change as compared to the Ser-60 -- Cyschange is consistent with the hypothesis that Ser-60 partic-ipates as a nucleophile in the cleavage reaction as has beensuggested for the corresponding Ser-119 of LexA (15). Noneof the umuD mutants tested completely eliminated the abilityof UmuD to function in mutagenesis. Although further workwill be required to establish whether these mutant UmuDproteins undergo cleavage but at a reduced rate and whetherthis accounts for the residual UV mutagenesis, the residualactivity could also be explained economically by suggestingthat uncleaved UmuD has some limited capacity to partici-pate in UV mutagenesis.

Cleaved UmuD Is Functional in Mutagenesis. To test moredirectly the hypothesis that cleavage of UmuD is importantfor mutagenesis, we constructed a umuD mutant of aumuD+,C- plasmid (pGW2020) in which overlapping termi-nation (TGA) and initiation (ATG) codons were introducedat the site in the umuD sequence that corresponds to theputative cleavage site. The plasmid (pGW2117) carrying thisengineered form of UmuD encodes two polypeptides ratherthan one (Table 1 and Fig. 1B). These two polypeptides arevirtually the same as those that would result from cleavage atthe Cys-Gly bond ofUmuD at positions 24 to 25. The smaller(NH2-terminal) polypeptide differs from the correspondingcleavage product in lacking the COOH-terminal cysteine.The larger (COOH-terminal) polypeptide is probably identi-cal to the corresponding cleavage product. Although itwould have an NH2-terminal formylmethionine when firstsynthesized, it is likely to be removed because NH2-terminalmethionines adjacent to glycines are removed by E. coli

FIG. 1. Pictorial representation of mutant-COOH UmuD proteins. (A) Mutant UmuD proteins

altered at the putative cleavage site (Cys-Gly atpositions 24 and 25) or the putative catalyticresidues (Ser-60 and Lys-97): Cys-24 -* Tyr(pLD1009); Gly-25 - Glu (pGW2110) or Lys(pGW2111); Ser-60 Ala (pGW2112) or Cys(pGW2113); Lys-97 -* Ala (pGW2115). (B)

-COOH pGW2117 encodes two polypeptides-i.e., asmall NH2-terminal and a large COOH-terminalpolypeptide of UmuD. (C) pGW2119 encodesthe NH2-terminal polypeptide. (D) pGW2122encodes the COOH-terminal polypeptide. (E)

-COOH Mutants that encode the two polypeptides andalso have Ser-60 -- Ala (pGW2118) or Lys-97-* Ala (pGW2121) changes. All plasmids de-picted in A are derivatives of the umuD+,C+

-COOH plasmid pGW2101 except for pLD1009, whichis a derivative of the umuD+,C+ plasmidpDS101. All plasmids in B-E are derivatives ofthe umuD+ plasmid pGW2020.

Biochemistry: Nohmi et al.

1818 Biochemistry: Nohmi et al.

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Proc. Natl. Acad. Sci. USA 85 (1988)

B

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FIG. 2. (A) Effect of plasmids encoding mutant UmuD proteinson UV mutagenesis in an AB1157 umuD44 strain (GW3200). o,pGW2101 (umuD',C+); A, pGW2110 (Gly-25 -* Glu); v, pGW2111(Gly-25 -. Lys); e, pLD1009 (Cys-24 -* Tyr); A, pGW2112 (Ser-60

Ala); c, pGW2115 (Lys-97 -- Ala); *, pGW2113 (Ser-60 -- Cys).(B) Effect of plasmids encoding both or either one of the NH2- orCOOH-terminal polypeptides of UmuD on UV mutagenesis in anAB1157 umuD44 strain (GW3200). o, pGW2020 (umuD+); *,pGW2117 (both polypeptides); A, pGW2122 (COOH-terminal poly-peptide); *, pGW2119 (NH2-terminal polypeptide) or without plas-mid.

methionine aminopeptidase with high efficiency (34). De-spite the absence of an obvious Shine-Delgarno sequence,we have been able to detect the synthesis of the largerpolypeptide corresponding to the COOH terminus of UmuDby the use of the maxicell technique (Fig. 3). When a plasmidcarrying this engineered umuD encoding two polypeptideswas introduced into a nonmutable umuD44 strain, it restoredthe UV mutability of the cell to that of a umuD+ strain (Fig.2B). This result strongly indicates that at least one of theproducts resulting from cleavage of the UmuD at its bondbetween Cys-24 and Gly-25 is capable of carrying out therole of the umuD gene product in mutagenesis. Furthermore,it rules out the possibility that the purpose of UmuD cleav-age is to inactivate UmuD.The COOH-Terminal Cleavage Product Is Necessary and

Sufficient for the Role of UmuD in Mutagenesis. A plasmid

2 3Bla _* - -31Kd

--21l.5K d

UmuD-.

UmuD

.J11 k'Ii.- -14.4Kd

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FIG. 3. IYS]Methionine-labeled proteins synthesized in maxi-cells containing pGW2020 or its derivatives. Lanes: 1, pGW2020(umuD+); 2, pGW2117 (encodes both UmuD polypeptides); 3,pGW2122 (encodes COOH-terminal polypeptide of UmuD, heredesignated UmuD*).

(pGW2119) that encoded only the polypeptide correspondingto the small NH2-terminal fragment of UmuD (Table 1 andFig. 1C) failed to complement the UV nonmutability of aumuD44 strain (Fig. 2B), whereas a plasmid (pGW2122) thatencoded only the large COOH-terminal polypeptide (Table 1and Fig. 1D) made the strain more UV mutable than aplasmid carrying umuD' (Fig. 2B). These results stronglysuggest that the COOH-terminal cleavage product of UmuDis both necessary and sufficient for the role of UmuD in UVmutagenesis. The COOH-terminal polypeptide of UmuD isexpressed at a higher level from the plasmid encoding onlythis polypeptide than from the plasmid encoding both theNH2- and COOH-terminal polypeptides (Fig. 3). This prob-ably accounts for the higher UV mutability of the straincarrying the plasmid encoding the COOH-terminal polypep-tide alone (Fig. 2B). We have not yet been able to rule outthe formal possibility that some NH2 terminal function ofUmuD is provided by the umuD44 gene on the chromosomebut consider this unlikely, since the COOH-terminal poly-peptide of UmuD is encoded on a multiple copy plasmid andsince increased expression of this COOH-terminal polypep-tide increases UV mutability.The COOH-Terminal Polypeptide of UmuD Restores UV

Mutability to recA430 Strains. To test the physiologicalsignificance of UmuD cleavage, we introduced plasmidscarrying either the engineered umuD encoding two polypep-tides or the COOH-terminal polypeptide of UmuD into aIexA71::TnS(Def) recA430 strain (3). The recA430 mutationhas differential effects on RecA's ability to mediate proteo-lytic cleavage. The RecA430 protein fails to mediate thecleavage of phage A repressor (35), mediates the cleavage ofLexA with reduced efficiency (22, 36-38), and mediates thecleavage of phage 480 repressor normally (17, 37). recA430strains are UV nonmutable. The introduction of the plasmidencoding the two engineered UmuD polypeptides partiallyrestored the UV mutability of this strain, whereas theplasmid encoding only the COOH-terminal UmuD polypep-tide restored the UV mutability of the strain to that of alexA71::Tn5(Def) recA+ strain carrying a umuD+ plasmid.We suggest that the more efficient restoration of mutabilityby the latter plasmid is due to a higher level of expression ofthe COOH-terminal polypeptide. The two plasmids alsorestored some UV mutability to a lexA + recA430 strain (datanot shown). The restoration of UV mutability to the recA430strains observed when we circumvented the need for UmuDcleavage strongly indicates that the primary cause for theUV nonmutability of recA430 derivatives is an inability tomediate the cleavage of UmuD. It furthermore implies thatthe purpose of RecA-mediated cleavage of UmuD is toactivate UmuD for its role in mutagenesis.A Third Role for RecA in Mutagenesis. In contrast to the

situation with the corresponding lexA71::TnS(Def) recA430strain, introduction of a plasmid encoding only the COOH-terminal polypeptide of UmuD into a lexA71: :TnS(Def)A(recA-srlR)306::TnIO strain did not suppress the UV non-mutability of this strain (Table 2). This observation raises thepossibility that RecA carries out a third role in mutagenesisbesides mediating the cleavage of LexA and UmuD.

Cleavage of UmuD Does Not Increase the SpontaneousReversion Frequency of argE3. The spontaneous reversionfrequency of argE3 in the recA+ umuD44 strains carryingplasmids encoding either the umuD encoding two polypep-tides (6.4 x 1O-') or the COOH-terminal UmuD polypep-tide (5.2 x 1O-') were virtually the same as a derivativecarrying a plasmid encoding the umuD+ gene (3.8 x 1O-').This contrasts with the highly increased spontaneous rever-sion frequencies for these alleles seen in strains in whichRecA has been activated genetically (21, 25). These obser-vations imply that the cleavage of UmuD by RecA* is not

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Proc. Natl. Acad. Sci. USA 85 (1988) 1819

Table 2. A plasmid encoding the COOH-terminal polypeptide ofUmuD does not suppress the UV nonmutability of a lexA(Def)A(recA) strain

Induced Arg+ revertants per

UV dose, survivorsJim2 GW6949* GW6964* GW6966*

0 0 0 02.5 0.6 <0.4 <0.25.0 1.5 <0.8 <0.67.5 2.0 <0.14 <0.2

UV mutagenesis was carried out as described (1) except that theirradiated cells were concentrated either 10-fold (for the samplesexposed to 0, 2.5, and 5.0 J/m2) or 100-fold (for the samples exposedto 7.5 J/m2) prior to plating. Colonies were counted after incubationfor 3 days at 370C.*GW6949 is GW2735 (31), a lexA7l::Tn5(Def) recA+ derivative ofGW1000 (10), containing pGW2020 (encodes UmuD+). GW6964 isGW6752, a lexA7J::Tn5(Def) A(recA-srlR)306::TnlO derivative ofGW1000, containing pGW2020 (encodes UmuD+). GW6966 isGW6752 containing pGW2122 (encodes the COOH-terminal poly-peptide of UmuD).

sufficient to account for the high spontaneous mutationfrequency observed at certain loci under such circumstancesand leaves open the question as to whether UmuD cleavageis necessary.

Differential Effects of Changing Ser-60 and Lys-97 in theCOOH-Terminal Polypeptide of UmuD. To explore furtherthe roles of Ser-60 and Lys-97 in UmuD function, weintroduced Ser-60 -+ Ala and Lys-97 -- Ala changes into theengineered umuD encoding two polypeptides (Table 1 andFig. 1E). The Ser-60 -+ Ala change only reduced the abilityof the COOH-terminal polypeptide of UmuD to function inmutagenesis by 50% (Fig. 4B). This contrasts with the majorreduction in mutagenesis observed when the same changewas introduced into a plasmid encoding an intact UmuDprotein (Fig. 2A) and suggests that the primary requirement

50

40

30

20

10

0 5 10 15 20

UV Dose (J/m2)

0

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FIG. 4. (A) Plasmids encoding the COOH-terminal polypeptideof UmuD restore UV mutability to a lexA71!:TnS(Def) recA430strain (GW6900). V, pGW2020 (umuD+); *, pGW2117 (encodesboth UmuD polypeptides); A, pGW2122 (encodes COOH-terminalpolypeptide of UmuD); o, GW2735 (IexA71::TnS recA +) carryingpGW2020 (umuD+). (B) Differential effects of changing Ser-60 andLys-97 in the COOH-terminal polypeptide of UmuD on UV muta-genesis in an AB1157 umuD44 strain (GW3200). *, pGW2117(encodes both UmuD polypeptides); A, pGW2118 (encodes bothUmuD polypeptides but with Ser-60 -* Ala); v, pGW2121 (encodesboth UmuD polypeptides but with Lys-97 -. Ala); *, AB1157umuD44.

for Ser-60 in UmuD function is for cleavage rather than forthe subsequent role of the COOH-terminal fragment in UVmutagenesis. In contrast, the Lys-97 Ala change caused asubstantial reduction in the amount of UV mutagenesisobserved (Fig. 4B), indicating that this change in some waydisrupts the function of the COOH-terminal fragment ofUmuD in UV mutagenesis.

DISCUSSIONThe results we have discussed in this paper strongly indicatethat RecA-mediated cleavage activates UmuD for its role inmutagenesis and that the COOH-terminal cleavage productis both necessary and sufficient for this role. Thus, it appearsthat RecA carries out two mechanistically related roles inUV and chemical mutagenesis: (i) transcriptional derepres-sion of the umuDC operon by mediating the cleavage ofLexA and (ii) posttranslation activation of UmuD by medi-ating its cleavage.The biological purpose of this regulatory system would

appear to be to give the cell an extra measure of control as towhether and to what extent it expresses the biochemicalactivities necessary for UV and chemical mutagenesis. Thisadditional regulatory feature adds to others that ensure thatUmuD and UmuC function is available only in emergencysituations: (i) the umuDC operon appears to require a rela-tively high degree of SOS expression to be fully induced (3,31), (ii) the UmuC protein is expressed at relatively low levelsdespite the strong transcription of the derepressed operon (1,10), and (iii) the UmuC protein has a relatively short half-life(L. Marsh and G.C.W., unpublished observations). Withrespect to future research in other organisms it is worth notingthat RecA-mediated cleavage of UmuD provides an exampleof an activating event induced by DNA damage that does notinvolve regulation at the transcriptional level (39).The failure of the engineered umuD encoding two poly-

peptides to suppress the nonmutability of a lexA(Def) ArecAstrain raises the possibility that RecA plays a third role inUV and chemical mutagenesis. However, this observationdoes not rule out the formal possibility that a single unre-

paired premutational lesion is lethal in E. coli recA strainsand that this accounts for the absence of induced mutants. Ifthere is a third role for RecA in mutagenesis, the nature ofthat role remains to be established. The observation ofTessman and co-workers (23, 25) that particular recA mu-

tants, which are constitutively activated for mediating pro-teolysis but are recombination-defective, are UV mutablesuggests that the third role cannot require all of the functionsof RecA required for homologous recombination. However,it might require a subset of these activities. Experiments byBridges and Woodgate (24) have argued that RecA caninfluence the misincorporation of an incorrect base opposite(at least) pyrimidine-dimer lesions. A possible explanationfor how this could occur is suggested by the observations ofFersht and Knill-Jones (40) and Lu et al. (41) that RecA caninhibit the 3'-) 5' exonuclease ofDNA polymerase III. Suchan inhibition of proofreading could facilitate misincorpora-tion opposite a noninformational or pseudoinformationallesions (3).The experiments of Bridges and Woodgate (24) also suggest

that UmuD and UmuC act after the actual misincorporation ofa base opposite a lesion, perhaps by facilitating the use of theterminus of the strand containing the misincorporated baseopposite the lesion as a primer for continued elongation.However, other mechanistic possibilities cannot be rigorouslyexcluded at this time. Whatever the biochemical function ofUmuD and UmuC proves to be, we suggest that it will involvea direct interaction between these two proteins or processedforms of these proteins. This suggestion is supported by the

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Biochemistry: Nohmi et al.

Proc. Natl. Acad. Sci. USA 85 (1988)

failure of the evolutionarily diverged umuD analog, mucA +,to complement a umuD mutant and by the failure of mucB+to complement umuC mutants (7). Furthermore, it is consist-ent with the apparently identical phenotypes of umuD andumuC mutants. It is possible that cleavage of UmuD in vivooccurs in a complex containing UmuC as well as UmuD andRecA and that it is the absence of purified UmuC that isresponsible for the relatively inefficient cleavage of purifiedUmuD by RecA in vitro (29). Once UmuD is cleaved, itsinteraction with UmuC could stimulate an inherent activity ofthe UmuC protein or create a new activity dependent on thepresence of both polypeptides.

Results presented in this paper suggest that the mecha-nism of UmuD cleavage is very similar to that of LexA andphage A repressor cleavage. Our finding complements thoseof Burckhardt et al. (29), which indicate that UmuD iscapable of conditional autodigestion and that the role ofRecA is to facilitate this self-processing. The self-processingof UmuD differs from that of LexA and A repressor in that aproduct of the self-processing events has a subsequentphysiological role. In this respect, however, it resemblescertain other self-processing reactions such as the self-splicing of tetrahymena ribosomal RNA (42). There arenumerous examples in biology of two proteins interactingand one protein becoming modified as a result of theinteraction. In the absence of evidence to the contrary, wetend to assume that the protein that becomes modified is apassive partner in the interaction and that the other proteinis an enzyme such as a protease or kinase. UmuD, LexA,and the phage repressors provide examples of situations inwhich the protein that becomes modified is capable ofconditional self-modification and the second protein facili-tates that self-modification. It should be interesting to seewhether conceptually similar strategies are used in otherinstances of protein modification.Our results also suggest that the primary role of Ser-60 of

UmuD is to function as a nucleophile in RecA-mediatedcleavage. As noted previously for LexA and phage A repres-sor (15), the sequences surrounding this seine, Gly-Asp-Ser-Gly for UmuD and Gly-Ser-Ser-Met for MucA, closelyresemble the consensus sequences around the active serinein mammalian serine proteases, Gly-Asp-Ser-Gly, and inmicrobial proteases such as subtilisin, Gly-Thr-Ser-Met. It ispossible that the mechanism of cleavage is even more closelyrelated to that of serine proteases than postulated by Slilatyand Little (15). In particular, it should be interesting to testwhether the acidic residue conserved between UmuD (Asp-68), MucA (Asp-69), LexA (Asp-127), and the phage repres-sors (aspartic residue in A and 480 and glutamic residue inP22 and 434) functions as a base to accept a proton fromlysine during RecA-mediated cleavage, just as Asp-102 ofchymotrypsin accepts a proton from His-57 during catalysis(43).

We thank Lorraine Marsh and the members of our research groupfor many helpful discussions. We thank Don Ennis for providing uswith the A(recA-srlR)306::TnlO mutation. This work was supportedby Public Health Service Grant CA21615 awarded by the NationalCancer Institute. J.R.B. was supported by a postdoctoral fellowshipfrom the American Cancer Society, Massachusetts Division.L.A.D. was a Jane Coffin Childs Postdoctoral Fellow.

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