DNA Structures Generated During Recombination Initiated By

(hpyright 0 1995 by the Genetics Society of America

DNA Structures Generated During Recombination Initiated by Mismatch Repair of UV-Irradiated Nonreplicating Phage DNAin Escherichia coli: Requirements for Helicase, Exonucleases, and RecF andRecBCD Functions

Wen-Yang Feng and John B. Hays

Department of Agricultural Chemistry, Oregon State University, Contallis, Oregon 97331 Manuscript receivedJanuary 31, 1995 Accepted for publication April 21, 1995

ABSTRACT During infection of homoimmune Escha'chia coli lysogens ("repressed infections"), undamaged nonreplicating X phage DNA circles undergo very little recombination. Prior UV irradiation of phages dramatically elevatesrecombinant frequencies, even in bacteria deficient in UvrABGmediated excision repair. Wepreviously reported that SO-SO% of thisUvrABC-independent recombination required MutHLS function and unmethylated d(GATC) sites,two hallmarks of methyl-directed mismatch repair. We now find that deficiencies in other mismatch-repair activities-UvrD helicase, exonuclease I, exonuclease VII, RecJ exonuclease-drastically reduce recombination. These effects of exonuclease deficiencies on recombination are greater than previously observed effects on mispair-provoked excision in vitro. This suggeststhat the exonucleases alsoplay other roles ingeneration and processing of recombinagenic DNA structures. Even though dsDNA breaks are thought to be highly recombinagenic, 60% of intracellular UV-irradiated phage DNA extracted from bacteria inwhich recombination is low-UvrD-, ExoI-, ExoVII-, or RecJ"disp1ays (near-)blunt-ended dsDNA ends (RecBCD-sensitive when deproteinized). In contrast, only bacteria showing high recombination (Mut+ UvrD+ Exo+) generate single- stranded regions in nonreplicating UV-irradiated DNA. Both recFand recBrecCmutations strikinglyreduce recombination (almost as much as a recF recB recC triple mutation), suggesting critical requirements for both RecF and RecBCD activity. The mismatch repair system may thus process UV-irradiated DNA so as to initiate more than one recombination pathway.

SCHERCHIA COLI contains versatile and efficient 1985).FENG et al. (1991)showed UvrA-independent E recombination systems; recombination-deficient recombinationto depend mostly on the E. coli mis- mutants (reviewed by CLARKand SANDLER1994) are match-repair activities MutH, MutL, and MutS, and on sensitive to UV light and other genotoxic agents, sug- the presence of unmethylated d(GATC) sequences in gesting that recombination mitigates the consequences the phage DNA. This suggested that methyl-directed of unrepaired DNA damage. Phage-A nonreplicating- mismatch repair sometimes converted photoproduct- DNA systems have been successfully used to analyze containing DNA into recombinagenic structures. damage-stimulatedrecombination in vivo ( LIN and MutH, MutL and MutS proteins mediate incision at HOWARD-FLANDERS1976; IKEDAand KOBAYASHI1978; unmethylated d(GATC) sequences near base-mispairs HAYSand BOEHMER 1978;PORTER et al. 1979; FENGet al. (Au et al. 1992). Subsequentexcision requires theUvrD 1991). The intracellular substrates in these experiments helicase(helicase 11) and, depending on the relative are well-defined 50-kb circles, in which DNA replication orientation of the mismatch andthe unmethylated and most transcription are blockedby (homoimmune) d(GATC) site, single-stranded-DNA exonucleases with phage repressor. Normally recombinationis negligible, 5' "f 3' (RecJ exonuclease, exonuclease VII) or 3' -+ 5' but UV irradiation of phages before infection (of unir- (exonuclease I) specificity (COOPERet al. 1993). Here radiated bacteria) increases recombinant frequencies we have asked whether MutHLSinitiated recombina- to 230% (HAYSand BOEHMER1978); stimulation is not tion of nonreplicating UV-irradiated phage DNA in due to induction of the E. coli SOS system (HAYSand UvrA- bacteria also requires these helicase and exo- LEE 1985). Irradiated phage DNA can give rise to re- nuclease activities. combinagenic structuresvia excision repair, but phages By analyzing phage DNA extractedfrom infected irradiated at moderate doses show substantial recombi- cells, we have further sought to determine whetherUV- nation even in UvrABC- bacteria (LIN and HOWARD- irradiated DNA is physically broken down by mismatch repair,whether any breakdown products resemble FLANDERS1976; PORTERet al. 1978; SMITHand HAYS structures thought to be recombinagenic, and whether mutations that affect recombination affect DNA break- Curresponding authm: John B. Hays, Department of Agricultural Chemistry, Oregon State University, ALS 1007, Cowallis, Oregon down.Repressed infections afford significant advan- 973313501. E-mail: [email protected] tages for study of recombination substrates and inter-

Genetic? 140: 1175- 1186 ( AuhWSt, 1995) 1176 W.-Y. HaysFeng and J. B. mediates in vivo; in the bacterial chromosome such by BODE (1965). Phages were radiolabeled, W irradiated, structures are typically infrequent,and concomitant and infected into lysogenic bacteria. Samples of unlabeled bacterial DNA plus ['Hlthymidine-labeled phage DNA,ex- DNA replication complicates analysis. tracted from repressed-infection bacteria, were mixed with Finally, we have asked whether this recombination of [ 14C]thymidine-labeled phage DNA that had been extracted UV-irradiated DNA conforms to models for separate from purified phage particles. Mixtures were layered directly RecF-dependent and RecBC(D)-dependent pathways. onto the topof 5-ml5-20% sucrose gradients and centrifuged Separate recombination pathways were originally pro- at 45,000 rpm in an SW50.1 rotor (Beckman)at 20" for 1.5 hr. Fractions (170 pl) were collected fromthe bottom.Aliquots of posed by CLARKand coworkers (HORIIand CLARK 40 pL were mixed with 3.5 mlof liquid-scintillation fluid (New 1973), to account for effects of certain mutations on England Nuclear "formula 989") and counted in a Beckman conjugal and transductional recombination. Because scintillation counter for 10 min. Spillover of 'H in the re&( C) mutants and recFmutants are each moderately channel was negligible. A setof 'IC samples spanning a range W sensitive, but recB(C) recF double mutants highly of 40-10,000 cpm was counted, and the rate of spillover of 'k cpm into the "H channel determined to be 16%. This sensitive, the separate-pathway hypothesis has been ex- correction was applied to 'H-cpm values. The DNA remaining tended to recombinational repair of UV-damagedchro- in each fraction was precipitated with EtOH, resuspended in mosomes. RecF is thought to assist recombinational fill- TE Buffer (10 mM Tris-HC1, 10 mM Nap EDTA, pH 8), and ing of daughter-strand gaps arising from photoproduct- used for other assays. blocked DNA replication, and the RecBCD nuclease/ Analysis of DNA with RecBCD enzyme: DNA in neutral- sucrose-sedimentation fractions, or pools of several adjoining helicase to carry out recombinational rejoining of dou- fractions (each sample contained 2300 cpm) was precipitated ble-strand breaks (GANESANand SEAWELL1975; SMITH by EtOH. Samples usually contained 2.5-7.5 ng phage DNA, and SHARMA1987; SMITHand WANG1989). plus a large excess of bacterial DNA.RecBCD enzyme was Here we find recombination to depend heavily on the kindly provided by Dr. ANDREW TAYLOR(TAYLOR and SMITH same helicase and exonuclease activities that mediate 1985). He assayed the specific activity to be -127,000 units per mg protein. Units are double-stranded exonuclease units repair of base-pair mismatches. The appearance of sin- of EICHLER andLEHMAN (1977):1 nmole of nucleotides solu- gle-stranded DNA (ssDNA), but not blunt-ended linear bilized in 20 min at 37"; estimated purity was greater than double-stranded (dsDNA), is positively correlated with 95% (by gel electrophoresis). Digestions were carried out as recombination. Both RecF and RecBC(D) function are described by TAYLOR and SMITH(1985), in a 20 pl solution required. containing 50 mM Tris-HC1 (pH 7.0), 1 mM dithiothreitol, 10 mM MgCI2, 200 mM ATP, 1 mg/ml bovine serum albumin, 2.5-7.5 ng of sample DNA plus -200 ng bacterial DNA, and MATERIALSAND METHODS two (or less, if indicated) units ofRecBCD enzyme. After Bacterial strains, bacteriophages, and plasmids: Bacterial incubation at 37" for 30 minutes, reactions were terminated strains, all E. coli K-12 derivatives, are described in Table 1. by addition of 0.2 volume of solution containing 200 mM Lambda bacteriophagesY1722, AAam32 Badplac5 red3~1857, EDTA, 0.1% sodium dodecyl sulfate, 50% sucrose, 0.2% bro- and Y1730, A plac5 lacZ118(oc) red3 61857,have been described mophenol blue, and0.2% xylene cyano]. Samples were mixed (FENGet al. 1991). Plasmid pCSR606 encodes the E. coli phrf with 0.1 volume of 3 M Na-Acetate, two volumes of chilled gene and some adjacent bacterial DNA (SANCARet al. 1984); 95% ethyl alcohol, and kept at -20" for 20 min. After centrifu- in pMS312, the E. coli mutp gene is inserted into the EcoRI- gation for 15 min at 13,000 rpm in a microfuge in a 4" room, nuclease-encoding gene of pSCC31 (SU and MODRICH 1986); pellets were dried and resuspended in 20 p1 TE. The amount pRC202 encodes the E. coli xseA+gene (CHASEet al. 1986); we of nonalcohol-precipitable radioactivity was determined both constructed pWFl by inserting the phage A cI (ind-) gene directly, as the fraction of DNA radioactivity remaining solu- (BglII fragment) into the BamHI site of the pACYC-derived ble, and as [1-(fraction precipitated)]. The average of these vector pAClacl9 (SCHAEFERand HAYS1990). two values was taken as the RecBCD sensitivity. Preparation and irradiationof bacteriophages: Stocks of X The resistance to RecBCD digestion of circular DNA du- bacteriophages, radiolabeled with ['HI thymidine, were pre- plexes containing gaps was verified using model substrates. pared and W-irradiatedessentially as described by FENGet al. Plasmid pBR322 was digested with restriction endonucleases (1991). UV-induced cyclobutane pyrimidine dimers (CPDs) EcoRI plus SalI (i), or BamHI plus SalI (ii),and the long were estimated on three different occasions, by extraction of fragments electrophoretically purified. These were annealed phage DNA and treatment with Micrococcus luteus or phage to pBR322 linearized by PstI endonuclease, to yield hydrogen- T4 UV endonuclease and sedimentation in alkaline sucrose, bonded circular heteroduplexes with (i) 0.65-kb or (ii) 0.28- as described by HAYS et al. (1985). Exposure to 20 J/m2 in- kb gaps, separated from nicks at the PstI site by 0.75 or 1.1 kb, duced -20 CPDs per A duplex; (6-4) photoproducts would respectively, plus linear homoduplexes. Gel electrophoresis be expected to be -15-30% as frequent as CPDs (FRANKLIN revealed putative gapped-circle bands migrating more slowly et al. 1982; MITCHELL1988). than the doublet correspondingto linear duplexes, but ahead Repressed infections and recombmation assays: Infection of presumed hydrogen-bonded concatamers trapped in the of E. coli bacteria lysogenic for A xis1 red3 c+(ind-), or bacteria wells. Under the digestion conditions used, all of the pre- carrying plasmid pWFl, with UV-irradiated homoimmune sumed linear species were removed after15 min, but the phages (XY1722 + AY1730 or AY1728 + x'Y1729; five of each putative gapped-circle bands showed no loss in intensity after parent per cell), incubation (with shaking) in broth at 38", 60 min RecBCD treatment (data not shown). These experi- harvesting of samples at various times, and extraction of DNA ments also rule out significant dsDNA endonuclease contami- for recombination and sedimentation assays, were essentially nation ofthe RecBCD preparations. as described (FENG et al. 1991). Analysis of DNA with mung-beanendonuclease: Mung- Sucrose gradient analyses: Zone sedimentation of DNA in bean endonuclease specifically cleaves single-stranded DNA sucrose gradients at neutral pH was essentially as described or RNA. 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HaysFeng and J. B. manufacturer (GibceBRL).Mixtures contained, in 0.5 ml, 10 mM Na-acetate (pH 5.0), 50 mM NaCl, 0.1 mM Zn-acetate, 1 mM cysteine, 5% (v/v) glycerol, 5-10 ng sample DNA plus -200 ng bacterial DNA, and 0.1 unit mung-bean endonuclease (1 unit of enzyme hydrolyzes 1 pg of denatured DNA to acid-soluble material in 1 min at 37”).The apparent requirement for excess endonuclease presumably reflects the presence of excess bacterial DNA and RNA in the reaction mixture. Linear A [‘4C]d~DNAincluded in reaction mixtures was not affected under these conditions. After 20 min incubation at 37”, reactions were terminated by addition of 0.2 ml TE buffer, and the DNA precipitated in EtOH. Preparation of partially single-stranded DNA by exonuclease 111 digestion: Digestion mixtures (1 ml) contained 0.4 mmol h DNA, 70 mmol Tris-HC1, pH 8, 1.7 mmol MgC12, 1 mmol EDTA and 1 mmol dithiothreitol. Reactions were carried out at37” for 10min, using 0.25-2.0 units of exonuclease INFECTION TIMEhid I11 per micromole of DNA (1 unit releases 10 nmol of acid- A 14 soluble nucleotide in 10 min at 37”). 0 B. Analysis of DNA by BND-cellulose chromatography: Ben- zoylated-napthoylated-DEAE-cellulose(BND-cellulose) was obtained from Sigma. A 25-g amount of resin was washed successively with 500 ml 2 M NaCl and 2 liters H20, and resuspended in 100 ml NET (0.3 M) buffer [0.001 M EDTA and $8 0.01 M Tris-HC1 (pH 7.5), containing 0.3 M NaCI]. Columns were formed by pouring 3 ml of the resuspended slurry sub- stance into a 3-ml syringe plugged with glass wool, and then washed with 10 ml of NET (0.3 M) buffer. DNA was loaded by slow passage (1 ml/min) of solution through the column. Columns were eluted with three 2-ml aliquots of NET (0.3 M) buffer, followed by six 2-ml aliquots NET (1 M) buffer. Elution was continued with an 0-2% gradient of caffeine dissolved in NET (IM)buffer, and then with 6 ml of 50% formamide 30 60 90 120 [in 2% caffeine and NET (1 M) buffer]. Twenty-one fractions INFECTION TIME(rnin) were collected; each was divided into several 0.5-ml aliquots and mixed with 5 ml of liquid scintillation fluid. The radioac- FIGURE 1.-Recombination in exonucleasedeficient and tivity of each fraction was calculated as the sum of the aliquot helicase-deficient mutants. Growth of hY1722 (AamBam lac+) radioactivities. and M730 (A+P lucZ118) phages to 20 J/m‘, infections of indicated uvrA(X) lysogens under room lighting, extraction of intracellular DNA at indicated times, electroporative trans- RESULTS fection of WF2872 (Aluc recA) bacteria, and measurementof the recombinant frequency (fraction of h A+B+ lac” among Recombination of W-irradiated nonreplicating phage total A’B’ infective centers) was as described under MATEM- ALS AND METHODS. Data correspond to average values for two DNA in UvrA-bacteria: We soughtto determine or three repressed infections, with half standard deviations whether activities that act in mismatch repair after indicated. (A) Single mutants. Strains infected: exo’( X), MutHLSmediated incision are also needed for recom- WF2860; xseA(A), WF3121; xseA(xseA+++)(A),WF3121- bination of nonreplicating UV-irradiated DNA. Mutant (pRC101); recJ(O), WF3122; xon(M), WF3119; uvrD( 6), derivatives of uvrA(h) bacteria, lacking helicase I1 SM2503. (B)Double mutants. Strains infected: exo+(X), WF2860; recJ((.), WF3122; xseA recJ (A), WF3124; xon xseA (uvrD), exonuclease I (xonA), exonuclease VI1 (xseA), (6),WF3123; xon recJ (W), WF3125. or RecJ exonuclease (recJ), were infected with W-irradiated X “Dam”’ phages [grown on bacteria unable to methylate d (GATC) sequences], and intracellular DNA plasmid fully corrected the recombination deficiency extracted and assayed forrecombination at various in AxseA bacteria (Figure lA, A). This appears to be times. In uurD bacteria recombination was only 1/10 to the first genetic evidence that ExoVII and E. coli exo- 1/4 of wild-type (exo”) values (Figure 1).We previously nuclease I (ExoI) can play positive roles in recombina- observed very similar reductions in mutH, mutL, and tion; previously, sbcB alleles of xonA, and xseA mutations, mutS bacteria (FENGet aZ. 1991). Recombination in exo- were found to actually increase bacterial conjugal and nuclease-deficient mutants-recJ xseA (ExoVI-) , or intrachromosomal recombination (KUSHNERet aZ. 1972; xonA (ExoI-) ”was reduced substantially, to 1/5 to 2/ CHASEand RICHARDSON 1977). In mutantslacking any 5 of exof frequencies (Figure 1: 0, A, respectively). two of the exonucleases, recombination was severely In previous studies of base-mismatch repair., in vitro, the reduced, andin AxonA recJ::Tn10 bacteria it was nearly repair defect in ExoVII- RecJ- extracts could not be abolished (Figure le). remedied by addition of purified E. coli exonuclease VI1 Physical analysis of nonreplicating W-irradiated (ExoVII) (COOPERet al. 1993). Here however, an xseA+ DNA processed by the mismatch-repair system: There Recombinagenic Mismatch Repair 1179

C. UV MutS*+ c

TUBE NUMBER FIGURE2.-Sedimentation and RecBCD-sensitivity of intracellular phage DNA. Growth of A Y1730 phages on Dam- bacteria (strain WF2873) with 'H- or l4C-thyrnidine, irradiation to 100 J/mz, infection of uvrA(A) lysogens with indicated additional phenotypes, extraction of intracellular DNA after 60 min, sedimentationin neutral sucrose, analyses of fractions for radioactivity, and of fractions (A)or pooled fractions (-) for sensitivity to RecBCD nuclease, were as described under MATERIALSAND METHODS. Sedimentation was right to left as shown. Radioactivity in individual fractions is expressed as percent of total ['HIDNA recovered from the gradient (0).Linear [I4C]DNAfrom A virions, added as a marker to all gradients is shown for each fraction in panel A (a),and the peak center (1) indicated in other panels. Some of the gradients shown here or in subsequent figures reflect small differences in sedimentation time and/or fraction size that cause the linearmarker to appear in different tubes. Representa- tive gradients from two or more experiments are presented. (A) Unirradiated phage in Mutt bacteria (strain WF2860), (I) apparent center at nicked-circle peak. (B) W-irradiated phage in Mut+ bacteria (strain WF2860). (C) UV-irradiated phage in MutSoverproducing (MutS+++)bacteria (strain WF2860 (pMS312)). (D) W-irradiated phagein MutS bacteria (strain WF2863). Total ['HIDNA recovered from gradients was 25,000-30,000 CPM; 600-6000 CPM were used for RecBCD analyses. have been relativelyfew studies ofDNA structures sucrose, along with exogenous linear X [ 14C] DNA.Unir- formedduring recombination in vivo; much of this radiated DNA (Figure 2A) was mostly (here 55%) in a work has focused on detection ofjoint molecules (see fast-sedimenting supercoiled form, aspreviously ob- for examples ANRAKU et al. 1969; IKEDAand KOBAYASHI served (BODE1965). A lesser portion (here 16%)sedi- 1978). Especially lacking have been analyses of poten- mentedat intermediate velocities, characteristic of tial recombinagenic substrates. In this initial physical nicked-circular and linear DNA. Although these latter study of the fate of nonreplicating UV-irradiated phage two species are not well resolved, the differences in X DNA in UvrA- Mut+ bacteria, we employed neutral- 3H:'4Cratios for tubes 18 and 19 us. 20 and 21 suggest sucrose sedimentation and treatment with various en- that for unirradiated DNA most of the central peak zymes, to semiquantitatively address three questions: consists of nicked-circular DNA (cross-hatched arrow), Does "mismatch repair" cause physical breakdown of which sediments 1.14 times faster than linear A DNA nonreplicating UV-irradiated DNA in vivo? Do any (filled arrow) (BODE1965). Only -9% of the radioac- breakdown products have properties associated with re- tivity appeared in the last three fractions, the position combinagenicity, e.g., double-strand ends or single- of very small DNA or nucleotides. The remaining 20% stranded regions? Do mutations that reducerecombina- was spread throughout the gradient. The amount of tion also alter the nature of the breakdown products? linear X [14C]DNA ineach fraction is shown here, so as Phage X ['HIDNA, extracted from bacteria after a to illustrate typical peak shape, but in subsequent pro- 60-min repressed infection, was sedimented in neutral files only the peak position is shown. 1180 W.-Y. Feng and J. B. Hays

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TUBE NUMBER TUBE NUMBER 3.-Sedimentation and RecBCD analyses of UV-irradiated phage DNA from exonuclease-or helicase-deficient bacteria. Growth and ‘H-thymidine labeling ofY1730 phages on Dam- bacierig (strain WF2873), irradiation of phages to 100 J/m‘, infection of uvrA(A) lysogens with indicated mutant phenotypes, extraction of intracellular DNA after 60 min, sedimentation in neutral sucrose together with linear “C-labeled A DNA (peak position, L), determination of’ percent of recovered ‘H CPM in individual fractions (A),and of proportion of’ RecBCD-digestible ‘H DNA in individual fractions (A)or pools of fractions (-), were as described under MI‘ER1AI.S AND METHOUS. RecBCD sensitivity corresponds to average between acid-soluble CPM and 1 - (fraction CPM precipitated). Representative gradients from three experiments are shown; RecBCD sensitivities varied by less than 235% among experiments. Total “H CPM recovered from gradients was 25,000-30,000; 1600-12,000 CPM were used to analyze RecBCDsensitivity. (A) m-irradiated phage in UvrD- bacteria(strain SM2503). (B) UV-irradiated phage in ExoI- bacteria(strain WF3119). (C) UV-irradiated phagein RecJ- bacteria(strain WF3122). (D) UV-irradiated phage in ExoVII- bacteria (strain WF3121).

“Dam”’ phages irradiated to 100 J/m‘ lose -80% somes would be expected to transfect poorly, account- of their biological activity within 40-60 min after (re- ing for thepreviously observed loss in biological activity pressed) infection of Mut+ UvrA- bacteria (FENGet al. (FENGet al. 1991). We deproteinized aliquots from 1991). Figure 2B shows that during this time all su- tubes (A) or pools of tubes (-) and determined the percoiled DNA is eliminated, and -25% degraded to fraction ofDNA in each tube that was digestible by very small fragments (last three tubes). Breakdown was RecBCD nuclease. Nearly 60% of the DNA from Mut+ more pronounced incells overproducing MutS protein Exo+ cells that sedimented at the velocity of linear X (Figure 2C): most of the products sedimented at the DNA appeared RecBCD-sensitive (Figure 2B). The cen- positions of full-length or slightly smaller linear X DNA, tral peak therefore appears to be a mixture of RecBCD- or very small DNA (now 33% of total). In MutS bacte- sensitive (near-) blunt-ended dsDNA, and RecBCD-re- ria (Figure 2D), most DNA appeared to sediment as sistant nicked-circular DNA and/or linear molecules supercoiled or nicked-circular DNA, but the resolution with overhanging ssDNA tails. Linear DNA with single- was not good enough to exclude linear products. stranded tails longer than 50 nt, or circular DNA with Linear dsDNA with blunt or near-blunt ends, a good large gaps, is known to be resistant to RecBCD digestion substrate for RecBCD helicase/nuclease (TAYLORand (PRELLand WACKERNAGEL1980), although excess en- SMITH1985), might be expected to initiate recombina- zyme is reported to slowly digest circular DNA with gaps tion by the RecBCD pathway. Also, linearized chromo- >650 nt (TAYL,ORand SMITH1985). We verified that Recombinagenic Mismatch Repair 1181 under our reaction conditions, model substrates containing 275- or 650-nt gaps were fully RecBCD-resistant A. ExoIII-treated (see MATERIALSAND METHODS). DNA from a MutS- infection showed no distinctive RecBCD-sensitive peak (Figure 2D). We cannot explain the significant back- ground of RecBCD sensitivityacross the gradientin this case; it might partially reflect breakage during manipu- lation of the fractions. The RecBCD nuclease preparation did not show any endonuclease activity in control reactions (see MATERIALS AND METHODS). DNA from bacteria lacking post-MutHLS mismatch- repair activities-UvrD helicase, exonuclease I, exonuclease 111, RecJ nuclease-was similarly analyzed by sedimentation and RecBCD treatment. In UvrD- Mut+ a bacteria (Figure 3A),loss of supercoiled DNA was pro- TUBE NUMBER nounced but incomplete. Perhaps in the absence of UvrD helicase, DNA ligase sometimes reseals nicks in- troduced by MutHLS, and gyrase reintroduces su- percoils. Very small DNA accounted for only 13% of cr; the total. DNA from exonuclease-deficient cells (Figure 0 B. ssDNA-endonuclease-treated 3, B-D) showed central peaks, at thepositions of linear caffeine-formamide pool DNA, that were narrower than those in EXO+ gradients 1M SALT 0-2% CAFFEINE FORMAMIDE (Figure 2B), and there was less very small DNA. About 60% of the (deproteinized) DNA from UvrD- or Exo- + bacteria (Figure 3) was RecBCD-sensitive, i.e., appeared to consist of blunt-ended linear dsDNA, even though recombination was severely reduced in these cases (Fig- ure 1). This suggests that intracellular linear dsDNA is not itself a sufficient substrate for recombination, because it lacks long ssDNA tails, or because its ends are protected by proteins, orfor other reasons. Al- though the apparent backgroundRecBCD sensitivity of the fastest-sedimenting DNA appeared as high as 30- 40% in the Exo- gradients, there was very little of this &I NUMBER TUBE DNA (Figure 3, A-C). FIGURE4.-BND-cellulose analysis of DNA standards. (A) We assayed for another structure expected to initiate Partially single-stranded A DNA. Growth of A Y1730 phages recombination, namely ssDNA, in two ways. First, we ana- with ['HI thymidine or ["C] thymidine radiolabeling, prepara- lyzed fractions from neutral sucrose gradients by chro- tion of phage DNA, treatment of ['HIDNA with exonuclease matography on BND-cellulose. Second, we exhaustively 111 to 10% ethanol solubility, and analysis by BND-cellulose treated aliquots of the fractions whose RecBCD digest- chromatography, were as described under MATERLALS AND ibility is shown in Figures 2, B and D, and 3, A-D with METHODS.A mixture of 3000 CPM (-300 ng) ExoIII-treated ['HI DNA ( W) plus untreated ['"C] DNA (0)was loaded onto mung-bean endonuclease to remove all ssDNA, both in a BND-cellulose column and eluted successively with 0.3 M tails and at interior gaps, and then digested any newly NaCl, 1 M NaCl, a 0-2% gradient of caffeine in 1 M NaCl, and formed blunt-endedlinear DNA with RecBCD nuclease. a solution of 50% formamide in 1 M NaCl plus 2% caffeine, The amount ofDNA previously resistant to RecBCD Typically 80-90% of loaded CPM was recovered in the tubes. nuclease, but now made susceptible, would correspond Data represent fraction of total recovered. (B) Analysis of caffeine-formamide DNA with mung-bean endonuclease and to the amount of DNA attached to ssDNA regions. The BND-cellulose. DNA fromfractions 14-25 from gradient actual amount of ssDNA might be much less. shown in A, mostly "-labeled (W) was precipitated in EtOH, For preliminary calibration experiments with BND- mixed with fresh untreated A [14C]DNA (0),and treatedwith cellulose, we digested A DNA (from phage particles) mung-bean endonuclease and rechromatographed on BND- to 10% ethanol solubility with exonuclease 111, which cellulose, as described under MATERIALS AND METHODS. selectively digests 5' "+ 3' at dsDNA ends (RICHARDSON et ul. 1964). A mixture of this partially single-stranded ssDNA (m). A gradient of 0-2% caffeine in 1 M NaCl [ 3H]DNA and untreatedlinear A [ I4C] DNA was applied eluted about half of the ssDNA, and a mixture of 50% to a BND-cellulose column, and eluted with a series of formamide, 1 M NaCl and 2%-caffeine eluted another solutions (Figure 4A). Elution with 1 M NaCl displaced fourth. About 1/10 of the dsDNA was eluted by caffeine most of the fully dsDNA (a),but virtually no partially but not by 1 M salt. This was the case even with rechro- 1182 W.-Y. Feng and J. B. Hays

I I I I 1 I 0 5 10 15 20 25 0 TUBE NUMBER v

FIGURE 5,"Single-stranded character of intracellular phage DNA. Growth and ["]thymidine labeling of Y1730 phages on Dam- bacteria (strain WF2873), irradiation of phages to 100 J/M', infection of uvrA(A) lysogene with indicated additional phenotypes, extraction of intracellular DNA after 60 min, sedimentation in neutral sucrose together with [I4C]thymidine-labeled A DNA (peak position, J), determinations of percent of recovered 'HCPM in individual tubes (0),of fractions of DNA in individual tubes (H) or pools of tubes (-) digestible by RecBCD nuclease after treatment with mung-bean endonuclease, and of fraction of DNA in individual tubes (0)or pools of tubes (I)retained by BND-cellulose in 1 M salt, were as described under MATERJALS AND METHODS. The data for treatment with mung-bean endonuclease plus RecBCD nuclease correspond to analyses of fractions from the gradients shown in Figure 2, B and D, and 3, A and D, respectively. These have been replotted at the positions of the corresponding fractions in A-D above, respectively, to facilitate comparisonof the two measurements of single- stranded character. matographed l-bf-salt-eluted material. DNA eluted by mentation analyses. We also measured, in aliquots of the caffeine-gradient andformamide steps,when the fractions shown in Figures2, B (Mut+UvrD'Exo') treated with ssDNA/RNA specific mung-bean endonu- and D (MutCUvrD-Exo+), and 3, D (Mut+UvrD+Exo- clease(Figure 4B, M), chromatographed identically VI-) and A (Mut+UvrD-Exo+), the extent to which with untreated dsDNA (0).The fraction of "caffeine- RecBCD sensitivity was increased by digestion with formamide DNA" therefore reflects the fraction with mung-beanssDNA-endonuclease. To facilitatecom- somesingle-stranded character. The BNDcellulose parison of the two measures of ssDNA character, we technique could notbe used to estimate the actual de- replotted the fraction-by-fraction mung-bean-endonu- gree of single-strandedness: samples digested with exo- clease/RecBCD-nuclease data obtained from the gra- nuclease I11 to varying extent showed similar chromato- dients shown in Figures 2, B and D, and 3, D and A, graphic properties (data not shown). at the positions of the corresponding fractions of the We fractionated DNA from repressed infections by gradients shown,respectively, in Figure 5, A-D, along neutral sucrose sedimentation and analyzed individual with the BND-cellulose data for these latter gradients. fractions or pools of fractions by BND-cellulose chro- In Mut'UvrD'Exo' bacteria, a significant fraction of matography (Figure 5). Comparison of the gradient material sedimenting more slowly than linear X DNA profiles in Figure5 with corresponding profiles inFig- (fractions 20-26) contained single-stranded regions,by ures 2 and 3 indicates the reproducibility of the sedi- both criteria. DNA corresponding to fractions 20-26 Recombinagenic Mismatch Repair 1183 DISCUSSION I uvrA mut+ (Darn- 20 J/m2 phage) - T I Experiments described here, together with our ear- lier work (FENGet al. 1991), implicate seven mismatch- repair activities-MutH, MutL, and Mu&, UvrD helicase, exonuclease I, exonuclease VII, and RecJ nuclease-in recombination of UV-irradiated nonrepli- catingd(GATC)-undermethylated phage A DNA in UvrA- bacteria, and in physical breakdown of irradiated DNA. This breakdown thus seems the likely cause of progressive loss ofbiological activity of the DNA in vivo (FENGet al. 1991). Breakdown products appear to in- clude lineardsDNA, evenin UvrD- or Exo- bacteria, in INFECTION TIME (min) which recombination is very low;in contrast,significant amounts of ssDNA appear only in recombination-pro- FIGURE6.-Effects of recF, recBC, and TecJmutations on re- ficient Mut+ UvrD' EXO+cells. The strong dependence combination. Growth of Dam- A Y1722 (Aum Bum lac+) and of this recombination on both RecF and RecBCD func- A Y1730 (A+PlucZ118) phages, irradiationto 20 J/mz, infections of indicated uvrA( A) repressorexpressing bacteria, ex- tion suggests that the operative pathway or pathways traction of intracellular DNA at indicated times, electropora- differ from those postulated for conjugal and transduc- tive transfection of A lac recA bacteria (strain WF2872), and tional recombination. measurementof A'B+ lac' among A+@ infectivecenters, The severe effects of uvrD, recJ xonA or xseA muta- were as describedunder MATERIALS AND METHODS. Data repre- tions onrecombination (Figure 1) pointto more sent averagesfrom two independent experiments,with ranges indicated. uwA (A) derivativesemployed: rec"(O), WF2905- than one function for therespective gene products, (pWF1); recJ(O), WF2908(pWF1); recF(U), WF2902(pWFl); perhaps involving more than one recombinagenic recBC(A),WF2916(pWF1); recFrecBC (O), WF29ll(pWF1). substrate. Possible routes to recombinagenic DNA structures via mismatch "repair" of UV-irradiated from a similar Mut+ UvrD+ Exo' gradient, after purifi- d(GATC)-unmethylated DNA are depicted in Figure cation by a second round of neutral-sucrose sedimenta- 7. MutHLS function is presumed to nick one or both tion, again chromatographed on BND-celluloseas if strands at fully unmethylated d(GATC) sites (Au et partially single stranded (data not shown). In contrast, al. 1992) [Figure 7, Step (l)]. UvrD helicase might MutS-, UvrD-, and ExoVI- bacteria yielded little or be required notonly to generate excision gaps [Fig- no ssDNA, by either criterion (Figure 5, B-D). RecJ- ure 7 (2A)], but also to prevent religation of near- and ExoI- bacteria showed patterns very similar to Exo- blunt dsDNA ends [Figure 7 (2B)], and perhapsfa- VI- patterns(data not shown).Thus, only in Mut+ cilitate resection of such endsby ssDNA-specific exo- UvrD+ EXO+bacteria did irradiated phages yield high nucleases [Figure 7 (4)]. recombinant frequencies (Figure l),and high ssDNA Mispair-protected excision in vitro requires only Ex01 levels (Figure 5A). if the d(GATC) is 5' to the mismatch [Figure 7 (2A)], Dependence of recombination on RecF and RecBCD or either ExoVII or RecJ exonuclease, if the d(GATC) functions: HORIIand CLARK(1973) hypothesized sepa- is 3' to the mismatch (not shown, for simplicity) (Coo- rate RecF- and RecBCD-dependent recombination PER et al. 1993; GMLLEYet al. 1993). If excision were the pathways. If the recombination initiated here by mis- only role for these exonucleases here, and d(GATC) match repair of UV-irradiated DNA proceeds only via sites 5' to photoproducts were as likely to be incised as a RecFdependent recombination pathway, or only via 3' sites, absence of ExoI, or of both ExoVII and RecJ, a RecBCD-dependent pathway, thenrecombination should therefore have reduced recombination only by

should be reducedby a recFmutation, but not arecB(C) one-half; absence of only one 5'"+ 3' exonuclease (Exo- mutation, orvice versa.If however, significant recombi- VI1 or RecJ) should have had even less effect. The nation takes place via each of two separate pathways, strong mutationeffects (Figure 1) thus suggest multiple recF or recB( C) mutations should have only partial ef- exonuclease roles. One ormore exonucleases may help fects and recF re&( C) mutations should completely prevent ligation of near-blunt dsDNA ends [Figure 7 eliminaterecombination. In fact, neitherprediction (2B)I and/or resect these ends to produce ssDNA over- was fulfilled: recombination appeared heavily depen- hangs [Figure 7 (4)]. Such overhangs might also be dent on both RecF and RecBCD functions (Figure 6). formed by endonucleolytic scission ofssDNA gaps Perhaps structures processed respectively by RecF and where repair synthesis was delayed [Figure 7 (3B and RecBCD are both involved in the same recombination 5)].The gaps might themselves be recombinagenic pathway, or distinct substrates initiate separate path- [Figure 7 (6A)l. A gap resulting from blockage by a ways that each require both RecF and RecBCD func- template photoproduct of repair synthesis [Figure 7 tion. (3A)I would be formally identical to a daughter-strand 1184 W.-Y. Feng and J. B. Hays

5’ CATC CTAG - (1) I MutHLS

I- (UvrD? Exonucleases?)

\ssDNA clease (RecBCD?) (3A) Exonucleases (8) ? PolIII - h 1

- ....~ - s sDNA endonuclease \ ( RecBCD? ) -” ....- FIGURE7.-Pathways for recombination initiatedby W photoproducts and MutHLS activity (see textfor discussion). MutHLS recognizes DNA in whicha UV photoproduct is near a fully unmethylated d(GATC) site (arbitrarilyshown 5’ to the photoproduct) and produces a nicked d(GATC) site (arbitrarily shownon the nonphotoproduct-containing strand) (l),which is converted by MutHLS into a dsDNA break (2B) or expanded into an excision gap (2A) by UvrD helicase + ExoI. dsDNA breaks might initiate recombination directly (8) or be resected by end-specific dsDNA exonucleases or helicase plus ssDNA exonucleases (4) to structures with ssDNA tails. Alternatively, PolIII might fill excision gaps DNAby synthesis (3A), until encounter with a photoproduct in the template strand, producing a partial-gap structure analogous to postulated daughter-strand gaps in semiconservatively replicating UV-irradiated DNA. Gapped structures might themselves initiate recombination (6A) or (6B), or be converted by endonucleases to ssDNA-tailed structures (3B, 5),which might then initiate recombination (7). gap in semiconservatively replicatingUV-irradiated combination pathway, as well as in initial MutHLScou- DNA. Such blocked-resynthesis gaps might be more re- pled excision. combinagenic than delayed-resynthesis gaps [Figure 7 The positive correlation of recombination with the pro- (6A) us. (6B)] if, for example, RecA protein were al- duction of ssDNA fits well with the requirement for an ready bound to photoproduct sites (Lu et al. 1986). ssDNA loading site for RecA in modelreactions (reviewed Recombinationmight also beinitiated by 5’- or 3’- by KOWALCZYKOWSKIet al. 1994), and with the postulated ended ssDNA tails [Figure 7 (’7)]. Inmodel “three- gapfilling role of recombination during replication of strand’’ recombination reactions between circular ssDNA Wdamaged DNA (RUPPand HowARDFLANDERs 1968). and linear dsDNA, reactions initiatedby 3”ssDNA ends, Surprisingly, (near-) bluntended &DNA, the substrate such as RecJ or ExoVII might produce by resection of for RecBCD helicase/nuclease, appears to be stable in linear dsDNA, appear favored (reviewed by KONFORTI UvrD- or Exo- mutants [note the paucity of smaller frag- and DAVIS1992). However, initiation by a 5”ssDNA end, ments inFigure 3, A-D) and nonrecombinagenic (Figure as ExoI might produce by resection, can also be signifi- l)]. Linear dsDNA in Exo- cells cannot have been pro- cantin certain circumstances(DUTREIX et al. 1991; tected against RecBCD simply by ssDNA tails (PRELLand KONFORTIand DAVIS1991). Furthermore, 5”ssDNA-ini- WACKENAGEL1980): mung-bean nuclease would have re- tiated three-strand reactions go efficiently to comple- moved the tails and increased RecBCDsensitivity, but this tion when the 3’-ended ssDNA displaced during branch was not observed in UvrD- or Exo- experiments (Figure migration is continuously digested by ExoI (KONFORTI 4, C and D). Perhaps the &DNA ends are protected in and DAVIS 1992; BEDALEet al. 1993). Exonucleases vivo by an unknown protein. might play analogous roles here. summary,In the severe The recombination seen here does not proceedby a reductions in recombination seen in Figure 1 suggest single pathway dependent on RecF but not RecBCD that UvrD helicase and/or one or more exonucleases function, or vice versa. This contrasts with evidence for may act at one or more points downstream in the re- separate RecF- and RecBCD-dependent pathways for Reco mb inagenic Mismatch Repair Mismatch Recombinagenic 1185 conjugal recombination and transduction (HORIIand nation: the pieces begin to fall into place. Crit. Rev. Microbiol. 20: 125-142. CLARK1973) and for recombinational toleration of un- COOPER,D. L., R. S. LAHUE and P. MODRICH,1993 Methyl-directed repaired DNAdamage (SMITHand SHARMA1987; SMITH mismatch repair is bidirectional. J. Biol. Chem. 268: 11823- and WANG 1989). The fact that recF and recB recC are 11829. DUTREIX,M., B. J. RAO and C. M. RADDING,1991 The effects on here each nearly as recombination-deficient as recF recB strand exchange of 5' versus 3' ends of single-stranded DNA in reccmutants arguesalso against roughly equal recombi- RecA nucleoprotein filaments. J. Mol. Biol. 219: 645-654. nant production by two independent pathways. Perhaps EICHLER,D. C., and I. R. LEHMAN,1977 On the role ofATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyri- both activities act in the same pathway. For example, bonuclease of Escherichia coli. J. Biol. Chem. 252 499-503. RecBCD helicase might open up (blunt-ended)dsDNA FENG,W.-Y., E. LEEand J. B. HAYS, 1991 Recombinagenic processing to produce ssDNA ends, and RecF facilitate recombina- of UV-light photoproducts in nonreplicating phage DNA by the Escherichia coli methyldirected mismatch repair system. Genetics tion of ssDNA gaps with these ends. This dual-substrate 129 1007-1020. model would explain as well the apparentfailure of the FISHEL,R. A., E. C. SIEGEI. and R. KOLODNER,1986 Gene conversion linear dsDNA produced in Exo- repressed infections in Escherichia coli. Resolution of hereroallelic mismatched nucleotides by cwrepair. J. Mol. Biol. 188: 147-157. to initiate recombination in the absence of ssDNA (Fig- FRANKLIN,W. A,, K M. Loand W. A. HASELTINE,1982 Alkaline ures 1, 4, and 5). lability of fluorescentphotoproducts produced in ultraviolet The semiquantitative correlation of recombination light-irradiated DNA. J. Biol. Chem. 257: 13535-13543. GANESAN,A. IC, and P. C. SEAWEIL,1975 The effectof kxA and rerF with the presence ofssDNA amongthe breakdown mutations on post-replication repair and DNA synthesis in E. coli products of UV-irradiated DNA, seen in this initial anal- K12. Mol. Gen. Genet. 141: 189-205. ysis, points the way to more refined studies of recombin- GRILLEY,M., J. GRIFFITHand P. MODRICH,1993 Bidirectional excision in methyldirected mismatch repair. J. Biol. Chem. 268: ing DNA in vivo. By adjusting the ratios of irradiated to 11830-11837. unirradiated DNA and the levels of rate-limiting activi- HAYS,J. B., and S. BOEHMER,1978 Antagonists of DNA gyrase inhibit ties, it should be possible to simultaneously maximize repair and recombination of W-irradiated phage X. Proc. Natl. Acad. Sci. USA 75: 4125-4129. measurable recombination and observable DNA inter- HAYS,J. B., and E. LEE, 1985 Repair and recombinationof non- mediates. Analyses of precursor-product relationships replicating UV-irradiated DNA in E. coli. 111. Enhancement of should then yield new insights into the structures of excision repair in W-treated bacteria. Mol. Gen. Genet. 201: 402-408. key recombination substrates. HAYS, J. B., S. J. MARTIN and K. BHATW,1985 Repair of nonreplicating UV-irradiated DNA Cooperative dark repair by Eschm'chia We thank ANDREW TAYLORand GERRVSMITH for generous gifts of coli Uvr and Phr functions. J. Bacteriol. 161: 602-608. RecBCD enzyme,JACKCHASE, SUSAN LOVETT, MARTIN MARINUS, PAUL HAYS,J. B., E. J. ACKERMANand Q. PANG,1990 Rapid and apparently MODRICH,GRAHAM WALKER, and BERNARDWEISS for providing bacte- error-prone excision repair of nonreplicating W-irradiated plas- rial strains and plasmids, TOM ELLENfor assisting with some experi- mids in Xenopus laeois oocytes. Mol. Cell. Biol. 10: 3505-3511. ments, and JUDITH HAYS for figures and manuscript preparation. HORII, Z.-I., and A. J. CIARK, 1973 Genetic analysis of the RecF MICHAEI. COX,ALVIN J. CIARK,STEPHEN KOWALCZYKOWSKI and PAUL pathway to genetic recombination in Escherichia coli K-12: isola- MODRICHmade helpful comments on the manuscript. This work was tion and characterization of mutants. J. Mol. Biol. 80: 327-344. IKEDA,H., and I. KOBAYASHI,1978 mA-mediated recombination of supported in part by pilotproject grants from the Oregon Unit, bacteriophage A: structure of recombinantand intermediate American Cancer Society, and the OSU Environmental Health Sci- DNA molecules and theirpackaging in vitro. Cold Spring Harbor ences Center (center grant ES00210). Symp. Quant. Biol. 43: 999-1008, KONFORTI,B. B., and R. W. DAVIS,1991 DNAsubstrate requirements for stable joint molecule formation by the RecA and SSB proteins LITERATURE CITED of Escherichia coli. J. Biol. Chem. 265: 6916-6920. KONFORTI,B. 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