Role of Saccharomyces Cerevisiae Msh2 and Msh3 Repair Proteins in Double-Strand Break-Induced Recombination

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 9214–9219, August 1997 Genetics

Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination

NEAL SUGAWARA*, FRE´DE´RIC PAˆQUES*, MO´NICA COLAIA´COVO, AND JAMES E. HABER†

Rosenstiel Center and Department of Biology, Brandeis University, Waltham, MA 02254-9110

Edited by Maurice S. Fox, Massachusetts Institute of Technology, Cambridge, MA, and approved June 18, 1997 (received for review May 20, 1997)

ABSTRACT When gene conversion is initiated by a dou- DNA containing small loops formed by frameshift mutations ble-strand break (DSB), any nonhomologous DNA that may be (16, 18). All of these mismatch repair events require the present at the ends must be removed before new DNA synthesis participation of the Pms1͞Mlh1 heterodimer (19, 20). Evi- can be initiated. In Saccharomyces cerevisiae, removal of non- dence that mismatch repair proteins might be involved in the homologous ends depends not only on the nucleotide excision removal of 3Ј ended single-strand DNA tails came from the repair endonuclease Rad1͞Rad10 but also on Msh2 and observations by Saparbaev et al. (21) that Msh2 and Msh3 were Msh3, two proteins that are required to correct mismatched required in spontaneous recombination events that also de- bp. These proteins have no effect when DSB ends are homol- pend on Rad1 and Rad10. It was not known whether these ogous to the donor, either in the kinetics of recombination or events were initiated by single-strand or double-strand DNA in the proportion of gene conversions associated with cross- lesions, nor at which molecular step Msh2 and Msh3 acted, but ing-over. A second DSB repair pathway, single-strand anneal- Saparbaev et al. suggested that these proteins might be in- ing also requires Rad1͞Rad10 and Msh2͞Msh3, but reveals a volved in the resolution of Holliday junctions. difference in their roles. When the flanking homologous We report here experiments showing that Msh2 and Msh3, regions that anneal are 205 bp, the requirement for Msh2͞ but not other mismatch repair proteins, are required to remove Msh3 is as great as for Rad1͞Rad10; but when the annealing nonhomologous DNA ends during both the initiation of gene partners are 1,170 bp, Msh2͞Msh3 have little effect, while conversion and the resolution of SSA intermediates that are Rad1͞Rad10 are still required. Mismatch repair proteins initiated by a DSB. When the ends of recombining DNA are Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and perfectly matched to their donor templates, Msh2 and Msh3 Msh3 recognize not only heteroduplex loops and mismatched are not required, nor do they appear to affect the resolution of -bp, but also branched DNA structures with a free 3؅ tail. Holliday junctions. Additional experiments support our con clusion that Msh2 and Msh3 act to facilitate Rad1͞Rad10- In Saccharomyces cerevisiae, homologous recombination initi- dependent removal of nonhomologous DNA ends. ated by double-strand breaks (DSBs) can occur by at least two distinct pathways: gene conversion and single-strand annealing MATERIALS AND METHODS (SSA) (1–6). In both cases, the ends of the DSB are resected bya5Јto 3Ј exonuclease to produce long 3Ј ended single-strand Strains and Growth Conditions. All strains were isogenic tails (7, 8). In gene conversion, these tails invade a homologous derivatives of either JKM111 (22) or tNR85 (23), both of which donor sequence and act as primers of new DNA synthesis. contain mutations of the HO cleavage site at MATa, so that However, for this to occur, any nonhomologous bases at the 3Ј only the introduced HO cutting site is cleaved. The two strains end must be removed, so that the primer end may basepair with are not isogenic; however we have found that HO-induced the donor template. Similarly, in SSA, complementary strands recombination of the same substrate (plasmid pJF5; ref. 1) of homologous regions flanking a DSB can anneal, producing yielded a nearly identical efficiency and kinetics of DSB repair an intermediate that has two nonhomologous 3Ј ended tails and the proportion of gene conversion events accompanied by that must be removed before new DNA synthesis and ligation crossing-over was the same (data not shown). Deletions of the can occur. In both instances, removal of nonhomologous tails RAD1, MSH2, and MSH3 were made by gene replacement (24) depends on the Rad1 and Rad10 proteins (9), which have been using the following constructs: rad1::LEU2 in plasmid pL962, shown in vitro to cleave 3Ј ended nonhomologous tails and from R. Keil; msh2::LEU2 in pRHB113 (25) and msh3::LEU2 which carry out a related function in nucleotide excision repair (26). G. Marsischky and R. Kolodner constructed and pro- (NER) (10–12). Other NER proteins are not required (13). vided strains RKY3111 (msh6::hisG) and RKY3112 In NER, Rad1͞Rad10 are presumably recruited to the site (msh3::hisG) in tNR85. The GAL::HO gene was integrated at of DNA damage by other proteins of the NER repair complex. the ADE3 locus (27) in JKM146 and was carried on plasmid We were interested in whether other proteins were required to pFH800 (28) in the tNR85 derivatives. Induction of HO attract Rad1͞Rad10 to the sites of strand invasion or strand endonuclease was accomplished by adding galactose (2% final annealing. One set of proteins that recognize DNA distortions concentration) to cells grown in lactate-containing medium at are the mismatch repair proteins, most notably Msh2, which 30°C, or by plating them on yeast extract͞peptone-galactose. has been shown to bind to mismatched bp (14), heteroduplex Measurement of DSB Repair Efficiency. To measure repair loops (14), and Holliday junctions (15). Msh2 has been shown and retention of pFP122, pFP140, and pFP120, cells were to form heterodimers with Msh6 (16, 17) and studies of the plated on yeast extract͞peptone͞dextrose and yeast extract͞ specificity of mismatch repair have led to the conclusion that peptone-galactose plates, and the resulting colonies replicated Msh2͞Msh6 primarily recognize and correct single bp mis- to plates lacking uracil, to determine plasmid retention. The matches, while Msh2 and Msh3 act to correct heteroduplex percent plasmid retention, a measure of successful gene con-

The publication costs of this article were defrayed in part by page charge This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: DSB, double-strand break; SSA, single-strand anneal- payment. This article must therefore be hereby marked ‘‘advertisement’’ in ing; NER, nucleotide excision repair. accordance with 18 U.S.C. §1734 solely to indicate this fact. *N.S. and F.P. contributed equally to this work. © 1997 by The National Academy of Sciences 0027-8424͞97͞949214-6$2.00͞0 †To whom reprint requests should be addressed. e-mail: haber@ PNAS is available online at http:͞͞www.pnas.org. hydra.rose.brandeis.edu.

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version, was calculated from the fraction of colonies retaining the plasmid on galactose medium divided by the same fraction on dextrose medium. Cell viability and deletion formation in the SSA assay were measured by plating cells on yeast extract͞ peptone͞dextrose and replica plating cells to synthetic complete medium lacking tryptophan (29) to score for the retention of pFH800. Recombination Substrates. Plasmids pFP120, pFP122, and pFP140 were similar to the centromeric URA3-marked plasmids containing inverted copies of the Escherichia coli LacZ sequence used previously (30), except that they did not carry LEU2. Their structure is shown in Fig. 1A. In pFP122, the recipient LacZ sequence contains a 40-bp MATa cleavage site. The donor locus carries a mutant cut site differing by only a single bp substitution at position Z6 that prevents HO cleavage (31). Therefore, the DSB generates a cut plasmid whose ends are almost exactly identical to the uncleaved donor sequence that is used to repair the break by gene conversion. In pFP140, the recipient LacZ sequence carries a 60-bp MATa cleavage site, so that the DSB creates two ends with 30 bp of nonhomology that must be removed before gene conversion can begin. pFP120 contains a 40-bp cleavage site in the recipient LacZ sequence, and the donor lacZ copy is deleted for the 878-bp ClaI–SacI fragment; consequently, the HO cleavage site of the recipient is now surrounded by two large sequences (308 and 610 bp) without any homologous counterpart in the donor. The SSA chromosomal substrates were derived from a triplication of URA3 sequences similar to tNS62 (8) except that FIG. 1. Effect of MSH and RAD genes on HO endonuclease- the distal ura3–52 gene was completely deleted and replaced induced gene conversion repair of a DSB. (A) Structure of the pFP122, by a THR4 gene. The resulting structure on chromosome V has pFP140, and pFP120 plasmids. (B) DSB repair efficiency in wild-type, a 205-bp region of the 5Ј end of the HindIII URA3 fragment rad1, msh2, msh3, msh2 rad1, and msh3 rad1 strains. duplicated on either side of an HO cleavage site. In Fig. 3 both duplicated regions were shown by sequencing to be identical to bp of the cut ends are not homologous to the intact donor. In the URA3 sequence derived from strain ϩD4 (32), except that wild-type strains, 69% of the cells complete recombination and they both possess the 5-bp deletion found in strain FL100 (32) retain the repaired plasmid. DSB repair is reduced 12-fold in andaTtoCsubstitution at nucleotide 57 of the ϩD4 sequence. the absence of Rad1 (Fig. 1B). These results are similar to In the strains shown in Fig. 4, the 205-bp segment to the left those reported in similar plasmids with a 117-bp HO recog- of the cleavage site is identical to that from FL100 and hence nition site (9). has seven single bp substitutions or frameshifts relative to the Efficient DSB repair also depends on Msh2 and Msh3. 205-bp region to the right of the cut site. In Fig. 5 similar Completion of HO induced gene conversion was reduced substrates with 205, 415, or 1,170 bp of identical sequences 6-fold in msh2 or 3-fold in a msh3 deletion (Fig. 1B). A rad1 were examined. Additional experiments were carried out using msh2 and a rad1 msh3 double mutant was reduced 7- to 10-fold. a LEU2-marked centromeric plasmid, pNSU208, similar to Similar results were found with plasmid pFP120 (Fig. 1A), with pJF6 (9), but carrying only 240 bp of directly repeated LacZ nonhomologous ends of 308 bp on one side and 610 bp on the sequences. other. msh2, msh3, and rad1 reduced gene conversion by 33-, DNA Analysis. DNA was extracted at intervals after HO 20-, and 23-fold, respectively, relative to a wild-type control induction as described (8) and appropriate restriction endo- (Fig. 1B). nuclease-digested DNA was analyzed on Southern blots. Gene In contrast, msh2, msh3, and rad1 had no effect when the conversions of plasmid pFP122 both with and without cross- donor also contained a (mutant) HO cut site and the HO- ing-over were analyzed by Southern blots probed with LacZ cleaved ends of the recipient were therefore homologous to the sequences (30). SSA was analyzed by probing with a HindIII– donor, except fora1bpdifference. A similar plasmid (pFP122; BamHI DNA fragment centromere proximal to the URA3 Fig. 1A) was constructed, where HO-cut ends are almost gene (8). The efficiency of SSA was measured by dividing the perfectly homologous to the donor DNA, by placing a 40-bp intensity of the product band after 5 hr of induction by the HO cut site in the recipient copy of LacZ and a mutant 40-bp intensity of the parental band prior to induction. Values were HO cutting site, differing by a single bp substitution, into the normalized to LEU2 or HIS3 sequences to control for the donor. Neither Rad1, Msh2, nor Msh3 was required for amount of total DNA per sample. efficient recombination (Fig. 1B). We conclude that Msh2 and Msh3 are required for the removal of nonhomologous tails in conjunction with the Rad1͞Rad10 endonuclease and are not RESULTS required when the DNA ends match their donor template. We MSH2 and MSH3 Are Required to Remove Nonhomologous have only used plasmid-borne substrates to analyze the re- Ends During Gene Conversion. The site-specific HO endonu- quirement for MSH2 and MSH3 on gene conversion. However, clease can be expressed under the control of a galactose- using chromosomal substrates we have also demonstrated that inducible promoter (33), to initiate DSB-mediated recombi- RAD1 is required when the ends are nonhomologous (data not nation in plasmid or chromosomal substrates containing an shown) and we presume that MSH2͞MSH3 would also be HO recognition site (31). We have examined gene conversion required for chromosomal events. in a centromeric plasmid where a 60-bp HO endonuclease Lack of Effect of MSH2 and RAD1 on Later Steps in Gene recognition site was introduced into one of a pair of inverted Conversion. Since rad1 and msh2 had little or no effect on repeated LacZ sequences (pFP140; Fig. 1A). The terminal 30 viability when the HO-cut ends were homologous to the donor, Downloaded by guest on September 26, 2021 9216 Genetics: Sugawara et al. Proc. Natl. Acad. Sci. USA 94 (1997)

we more closely examined these strains by Southern blots of DNA extracted at intervals after HO induction, to discern whether these mutations had an effect on the kinetics of recombination or on the proportion of gene conversion events associated with crossing-over. Southern blots for pFP122 in wild-type, msh2, and rad1 strains (Fig. 2) show the kinetics of appearance and disappearance of HO-cleaved DNA fragments and the time of appearance of the two PstI restriction fragments representing gene conversions associated with crossing- over. PhosphorImager analysis of these Southern blots showed that the proportion of gene conversions associated with crossing-over was Ϸ23.5% in the wild-type strain. This proportion was not significantly affected by msh2 (19%) or rad1 (20.5%). Moreover, there was no change in the kinetics of appearance of the crossover products in the msh2 or rad1 strains. Thus, Msh2 and Rad1 are required at the same step: to remove nonhomologous single-stranded DNA from the end of recombining DNA. Neither msh2 nor rad1 affects any of the other steps that should be common to all gene conversion events (e.g., DNA pairing, strand invasion per se, branch migration, or Holliday junction resolution). Role of Mismatch Repair Genes in SSA. When a DSB is flanked by homologous regions, repair can occur by nonreciprocal recombination leading to a deletion. Most of these events arise by SSA (Fig. 3A) but could also occur by ‘‘one- ended’’ recombination events, in which one DNA end could invade the other flanking homologous region before it became single-stranded (1, 9). In contrast to efficient deletion formation between 205-bp URA3 sequences in wild-type cells (Fig. 3B), deletions were greatly reduced in a rad1 derivative (Fig. 3B). The effect of msh2 and msh3 deletion mutations was very similar to rad1 (Fig. 3B). Cell viability (those that repaired the DSB) went from 77% in the wild type to 3.7% in msh2 and 5.4% in msh3, very similar to a rad1⌬ strain (1.0%). Similar experiments were carried out with a deletion of MSH6, whose gene product has been shown to form heterodimers with Msh2, but which has been implicated princi- pally in the repair of single bp substitutions rather than small loops in heteroduplex DNA (16–18). In these experiments (Fig. 3C), SSA was monitored on a centromeric plasmid containing 240 bp of LacZ homology, in direct orientation, flanking the DSB, similar to plasmids described (1, 9). As shown in Fig. 3C, msh6 does not impair deletion formation by SSA, while msh3 does. A similar result has been obtained using chromosomal substrates identical to those shown in Fig. 3A (data not shown). This result argues that Msh2͞Msh3 recognizes nonhomologous tails during recombination in a fashion similar to their recognition of insertion͞deletion heterologies, whereas Msh2͞Msh6, which mainly recognizes bp substitutions, plays no significant role in SSA. Mismatch repair of bp substitutions and frameshifts requires not only Msh2, Msh3, and Msh6, but also the Mlh1 and Pms1 proteins (19, 20). However, while both MSH2 and MSH3 are required to remove nonhomologous DNA tails, PMS1 and MLH1 are not (Fig. 4). We first showed this for pms1 when the 205-bp repeats were perfectly homologous (data not shown).

combination in wild-type (B), msh2 (C), and rad1 (D) strains exhibit the appearance of two crossover products one hr after HO cleavage of the 5.2-kb PstI LacZ fragment. One of the two gene conversion products comigrates with the 5.2-kb parental fragment. However, in these strains carrying an integrated ADE3::HO gene, HO cleaves essentially all of the plasmid targets (data not shown), so that virtually all of the 5.2-kb band remaining at the end of the experiment represents gene conversions without crossing-over and not the original FIG. 2. Kinetics of HO-induced crossing-over. (A) When pFP122 parental fragment. The sum of the intensities of the 4.3- and 8.5-kb undergoes HO cleavage, gene conversions both with and without crossover bands divided by the sum of the crossover bands and the 5.2- crossing-over arise. The crossover products can be distinguished on and 8.3-kb noncrossover bands, measured 24 hr after HO induction, Southern blots, probed with LacZ sequences, by the sizes of PstI- indicates the proportion of gene conversions associated with crossing- restriction fragments homologous to LacZ (1, 10). HO-induced re- over. Downloaded by guest on September 26, 2021 Genetics: Sugawara et al. Proc. Natl. Acad. Sci. USA 94 (1997) 9217

formation suggesting that (i) they are not required to excise nonhomologous tails and (ii) they are not responsible for the discouragement of annealing by 3% heterology. In contrast, the absence of msh2 caused a dramatic decrease in deletion formation (Fig. 4). These results argue that MSH2 and MSH3 play a role in the removal of nonhomologous tails from the annealed intermediate that is distinct from their roles in mismatch correction or heteroduplex DNA formation. What- ever improvement msh2 might have caused in increasing recombination between diverged substrates was overwhelmed by its very strong negative effect on the completion of SSA. Distinguishing the Roles of RAD1 and MSH2͞MSH3 During SSA. Msh2 and Msh3 might act in several different ways in removing nonhomologous DNA tails. Msh2͞Msh3 may recognize and bind to branched intermediates and present them to Rad1͞Rad10 for cleavage. They might also stabilize initially unstable strand annealing intermediates, allowing Rad1͞ Rad10 more time to act. Finally they might form a complex with Rad1͞Rad10 to increase its endonuclease activity. Fur- ther experiments have shown that the requirement for Msh2 and Msh3 in SSA depends on the length of the regions that anneal. Three SSA substrates were constructed on chromosome V in which the lengths of the single-stranded DNA tails that must be removed remained constant, but the length of the annealed region increased from 205 bp to 415 bp or to 1,170 bp (Fig. 5). The absence of RAD1 had the same severe effect on deletion formation, regardless of the length of the flanking homologous regions. In contrast, while the requirement for MSH2 and MSH3 was as great as for RAD1 with 205-bp flanking regions, the absence of the mismatch repair proteins was less profound with longer flanking regions. With 1,170-bp flanking homology, msh2 and msh3 reduced SSA only 25% relative to wild type. The significance of this difference will be discussed below.

DISCUSSION We propose that the Msh2͞Msh3 heterodimer recognizes and binds to a branched DNA structure such as that formed by strand invasion or strand annealing. Msh2͞Msh3 either (i) stabilizes this structure so that Rad1͞Rad10 can trim off the nonhomologous tail, and͞or (ii) directly participates in recruit- ing Rad1͞Rad10 to the sites of recombination, in the way that FIG. 3. Requirement for Msh2, Msh3, and Rad1 in SSA. (A) SSA other NER proteins may recruit Rad1͞Rad10 to the sites of between identical 205-bp URA3 gene segments flanking an HO- DNA photodamage. induced DSB. Extensive 5Ј to 3Ј exonuclease digestion produces 3Ј Interestingly, the requirements for Msh2͞Msh3 are different ended DNA tails in which complementary regions can anneal. Cleav- during the initiation of gene conversion and the removal of 3Ј age of the 3Ј-ended tails allows new DNA synthesis to fill in gaps and ended tails during SSA. In SSA, Msh2 and Msh3 are less ligate the deletion. Diagnostic BglII fragments are indicated. (B) important as the length of the flanking homologous regions Southern blot of the time course of deletion formation in wild-type cells. BglII-digested DNA reveals HO cleavage and the formation of increases from 205 bp to 1 kb. In contrast, Msh2 and Msh3 are the deletion in the wild-type strain (tNS1379). Deletion formation is as necessary as Rad1 during gene conversion, even though the significantly reduced in msh2 (tNS1406), msh3 (tNS1390), and rad1 lengths of the homologous regions shared between the donor (tNS1429) strains. SSA was analyzed by probing with a HindIII– and recipient are Ϸ2 kb on either side of the DSB. We believe BamHI DNA fragment centromere proximal to the URA3 gene (8). this difference reflects the nature of the intermediate struc- (C) Southern blot of the time course of deletion formation on plasmid tures on which Rad1͞Rad10 acts in SSA and in gene conver- pNSU208 carrying 240 bp of directly repeated LacZ sequences in sion. In gene conversion, the initial encounter between the wild-type cells (tNS865), compared with msh3 (tRKY3112) and msh6 invading strand and the target duplex DNA is a relatively (tRKY3111) strains. DNA was digested with EcoRI. All strains were unstable paranemic joint that cannot be converted into a more derivatives of tNR85 (22) and carried GAL::HO on a TRP1-marked plasmid, pFH800 (27). stable plectonemic configuration until the 3Ј ended tail is excised (35). We suggest that such unstable intermediates require the presence of Msh2͞Msh3 to facilitate Rad1͞Rad10 It was then of interest to know if SSA would be differently cleavage. In contrast, annealing between two single-strand tails affected if the two repeats were divergent, since previous can intertwine more readily to form plectonemic joints, though studies had shown that homologous recombination was im- initial encounters could also be via paranemic associations. It proved by mutations in msh2, msh3, and pms1 (25, 26, 34). We appears that the stability of these interactions significantly therefore examined SSA in substrates that carried seven increases between 0.2–1 kb of homology and that Msh2͞Msh3 heterologies in the 205-bp chromosomal regions flanking the are especially critical when the homologous regions are short. DSB. SSA was reduced in the wild-type strain to Ϸ25% of the These results argue that Msh2͞Msh3 recognize branched value for fully homologous sequences (Fig. 3B vs. Fig. 4). structures and stabilize them, thus enabling Rad1͞Rad10 to Neither pms1 nor mlh1 affected the efficiency of deletion cleave off the tails. Downloaded by guest on September 26, 2021 9218 Genetics: Sugawara et al. Proc. Natl. Acad. Sci. USA 94 (1997)

FIG. 4. Effect of PMS1 and MLH1 on SSA. An experiment similar to that of Fig. 3 was performed except that the 205-bp repeats flanking the DSB differed by seven heterologies (see Materials and Methods). Strains included wild type (tNS1357), mlh1 (tNS1396), pms1 (tNS1394), and msh2 (tNS1359).

These data provide the first direct evidence of the specific homologous tail and thus require RAD1 and MSH2. Second, steps in recombination that require Msh2 and Msh3. Previ- it must be noted that previous studies have shown that ously Saparbaev et al. (21) had reported that MSH2 and MSH3 recombination in a rad52 strain between the same his4 alleles are required in a RAD1–RAD10-dependent spontaneous re- used by Saparbaev et al. does not actually occur by reciprocal combination, either in deletion formation (which we presume exchange; instead it occurs by a nonreciprocal event in which occurred by SSA) or in a RAD52-independent pathway for only one of the two participating chromatids is recovered (37). spontaneous recombination involving unequal sister-chroma- The actual mechanism, which might involve the invasion of tid crossing-over. They suggested that Msh2 and Msh3 proteins only one DNA end (e.g., ref. 38), remains unknown. It is of might aid the resolution of Holliday junctions. However, our course possible that Msh2 and Msh3 play still other roles in data demonstrate that DSB-initiated gene conversions are not spontaneous recombination, different from those we observe impeded by a msh2 mutant, so long as the invading strands for DSB-mediated recombination. However, RAD52- have homologous ends. These conversions occur both with and dependent spontaneous recombination between homologous substrates that occur by a true reciprocal crossing-over are not without crossing-over (and presumably involve Holliday junc- affected by msh2 or msh3 (26, 34). tions, ref. 36). Why then should the events studied by Sapar- Our results imply that Msh2 and Msh3 recognize a branched baev et al. (21) be rad1- and msh2-sensitive, since the two sister DNA structure with a free 3Ј end. This structure is different chromatids are identical and should have perfect homology? from all previously reported interactions of Msh2 and Msh3 First, if the lesions initiating these events (either DSBs or with DNA, where these proteins bound to mismatches or single-strand nicks) occurred in the interval between the re- heteroduplex containing loops in continuous duplex strands of peated sequences, then unequal sister recombination between DNA, in vitro (14) and apparently in vivo (39). A recent report the misaligned repeats would also require removal of a non- also shows that Msh2 can, by itself, bind to another branched structure, the Holliday junction (15); however, our data do not support suggestions (15, 25) that the absence of Msh2 would affect the resolution of this structure and hence the proportion of gene conversions that are resolved with crossing-over. Still, Msh2 might affect the extent of branch migration (40) in a way that our assays do not detect. The key point of our study is that MSH2 and MSH3, but not MSH6 nor PMS1 nor MLH1, play a role in removing nonhomology from the ends of recombining DNA. Thus, the removal of nonhomologous tails to initiate new DNA synthesis during recombination brings together an endonuclease from the NER pathway with heterology-recog- nizing proteins from the mismatch repair pathway, to carry out a novel role in recombination.

G. Marsischky and Richard Kolodner generously constructed and provided msh6 and msh3 strains. We thank R. Keil, R. Lahue, and G. Crouse for plasmids. Susan Lovett, Bernard Lopez, and members of the Haber lab made helpful comments on the manuscript. This work FIG. 5. Effect of length of the annealed region on requirements for was supported by National Institutes of Health Grant GM20056. M.C. RAD and MSH genes during SSA. Experiments identical to those was supported by U.S. Public Health Service Training Grant in shown in Fig. 3 were carried out with SSA substrates with different Genetics GM 01722 and National Institutes of Health Minority lengths of homology flanking the HO-induced DSB. Strains were Predoctoral Fellowship GM18050. F.P. is a fellow of the Jane Coffin identical to those used in Fig. 3, whose data are plotted here for the Childs Memorial Fund for Medical Research. experiment with a length of 205 bp. The lengths of the nonhomologous tails to be excised were the same in all cases. Data for rad1, msh2, and 1. Fishman-Lobell, J. & Haber, J. E. (1992) Mol. Cell. Biol. 12, msh3 were normalized to the wild-type value at each point. 1292–1303. Downloaded by guest on September 26, 2021 Genetics: Sugawara et al. Proc. Natl. Acad. Sci. USA 94 (1997) 9219

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