The Chromosome Bias of Misincorporations During Double-Strand Break Repair Is Not Altered in Mismatch Repair–Defective Strains of Saccharomyces Cerevisiae

The Chromosome Bias of Misincorporations During Double-Strand Break Repair Is Not Altered in Mismatch Repair–Defective Strains of Saccharomyces cerevisiae

Carolyn B. McGill, Susan L. Holbeck and Jeffrey N. Strathern Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute–Frederick Cancer Research and Development Center, ABL–Basic Research Program, Frederick, Maryland 21702-1201

ABSTRACT Recombinational repair of a site-speciﬁc, double-strand DNA break (DSB) results in increased reversion frequency for nearby mutations. Although some models for DSB repair predict that newly synthesized DNA will be inherited equally by both the originally broken chromosome and the chromosome that served as a template, the DNA synthesis errors are almost exclusively found on the chromosome that had the original DSB (introduced by the HO endonuclease). To determine whether mismatch repair acts on the template chromosome in a directed fashion to restore mismatches to the initial sequence, these experiments were repeated in mismatch repair-defective (pms1, mlh1, and msh2) backgrounds. The results suggest that mismatch repair is not responsible for the observed bias.

EVERAL years ago, Drake (1994) reviewed the first The repair of double-strand DNA breaks (DSB) in S 75 years of research on the origin of mutations and yeast occurs predominantly by recombinational mecha- made predictions of what would be discovered in the nisms that involve DNA synthesis. This recombination- early twenty-first century. While the progress in basic associated repair synthesis provides an additional oppor- research in this field has been impressive, and the prag- tunity for replication errors. Indeed, repair of a site- matic consequences of defects in processes that limit specific DSB induced by HO endonuclease, which cuts replication fidelity are increasingly apparent, he high- only once in the yeast genome, causes a several 100- lighted the multiple areas influencing the origin of fold increase in the reversion rate of a nearby (0.3-kb) spontaneous and induced mutations that are not yet marker (Strathern et al. 1995). It has been demon- understood. It is encouraging to see that the progress strated that the mutation rate is elevated in meiosis he predicts will be made on the multiple issues that are (Magni and von Borstel 1962) and that such muta- still unresolved. Thus far, two main pathways for the tions are positively correlated with nearby crossover origin of base substitution mutations have been estab- events (Magni 1964). A correlation of spontaneous mu- lished: alterations of the template (including those tation and mitotic recombination leading to crossovers caused by chemical modification and irradiation) re- has also been demonstrated (Esposito and Bruschi sulting in defective coding capacity, and misincorpora- 1993). Combined, these observations suggest that ele- tions on undamaged templates during replicative syn- vated DNA synthesis error rates may be a general feature thesis. Chemical alterations of the template are often of recombination processes. Perhaps the elevated muta- viewed as premutagenic lesions because they are subject tion rate associated with recombination contributes sig- to various repair pathways that can remove or reverse nificantly to the overall mutation rate. We demonstrated such lesions (Friedberg 1988). Similarly, the initial mis- that the DNA synthesis associated with DSB repair has incorporations by DNA polymerase should be consid- a relatively high mutation rate (Strathern et al. 1995). ered premutagenic. Elegant studies led to the view that Our current work focuses on the relative contributions net base substitution rates reflect the combined conse- of polymerase errors and escape from directed mis- quences of initial misincorporation, followed by editing match repair to these recombination-associated errors. via 3Ј–5Ј exonuclease activity of the DNA polymerases The translesion polymerase encoded by REV3 (Nelson and the directed mismatch repair processes (Kunkel et al. 1996) is responsible for most of the base substitu- and Loeb 1981; Kornberg and Baker 1991; Beckman tion errors during DSB repair (Holbeck and Strath- and Loeb 1993; Schaaper 1993). We are studying the ern 1997). In this report, we investigate the contribution relative effects of these processes on the fidelity of DNA of mismatch repair to the rate and distribution of DNA synthesis during recombination. misincorporations during DSB repair. In the experiments demonstrating that there is an elevated mutation rate associated with DSB-initiated mi- Corresponding author: Jeffrey N. Strathern, Gene Regulation and totic recombination, we noted that the mutations are Chromosome Biology Laboratory, NCI–Frederick Cancer Research and Development Center, ABL–Basic Research Program, Box B, Bldg. nonrandomly distributed between the two interacting 539, Frederick, MD 21702-1201. E-mail: [email protected] chromosomes (Strathern et al. 1995). Because only

Genetics 148: 1525–1533 (April, 1998) 1526 C. B. McGill, S. L. Holbeck and J. N. Strathern

models for this bias. In one class of models, the noncrossover events are the result of mechanisms in which the newly synthesized DNA, and hence all the errors, are inherited by the cut chromosome. All the newly synthesized DNA is inherited by the repaired chromosome if a double Holliday junction intermediate is re- solved by a topoisomerase (Figure 2B; Nasmyth 1982; Thaler et al. 1987; Hastings 1988; McGill et al. 1989) rather than by strand cleavage or if replication proceeds by a conservative mechanism (Figure 2C). An alternative model for the bias in inheritance of errors is that resolution proceeds by cleavage of Holliday junctions. This allows intermediates carrying errors on either chromosome, but the correction of mismatches on the template chromosome is biased toward restoration, while correction of the mismatches on the repaired chromosome is random. We postulated that if mismatch correction were directed by nicks, a different distribution of nicks on the two chromosomes would promote the observed bias (Strathern et al. 1995). Specifically, we noted that resolution by cleavage of the two Holliday junctions to produce a noncrossover DSB repair results in the initially uncut (template) chromosome having one original strand with no nicks and a second strand Figure 1.—DSB repair model applied to the reversion of carrying any newly synthesized DNA that would include trp1-488. (A) Physical description of the TRP1-HIS3 interval. strand discontinuities (Figure 1, D and E). The presence The TRP1 and HIS3 genes were inserted into an EcoRI site of the nicks on the strand with the newly synthesized centromere proximal to MAT. Mutations in the HIS3 gene were made by filling in the NdeI site at codon 64 (his3-192) DNA (and hence any new misincorporations) might be or by filling in the AspI site at codon 207 (his3-621). Both signals for the directed repair of mismatches, as has MAT alleles (MAT␣-inc and MATa-inc) are resistant to cutting been demonstrated for Escherichia coli mutS system (La¨n- by HO endonuclease. The HO site was inserted into a poly- gle-Rouault et al. 1987; Lahue et al. 1989; Modrich linker between trp1 and his3 on the cry1 MATa-inc chromo- 1991) and for eukaryotes (Holmes et al. 1990: Thomas some. Both chromosomes carry the trp1-488 mutation. The trp1-488 allele was made by site-directed oligonucleotide muta- et al. 1991). This would be true for the template chromo- genesis to change two bases and create an SpeI site (from some for either pairing of cleavages of the two Holliday ACTGGG to ACTAGT) with an in-frame stop codon at codon junctions that would lead to a noncrossover (Figure 1, 163 (trp1-488). (B) The DNA strands of the broken chro- D vs. E). In contrast, the DNA of the chromosome that mosome are white and are subject to recision. The uncut originally had the DSB would have nicks on both strands chromosome used as a template for repair is grey. (C) Newly synthesized DNA, copied from the uncut chromosome, is and hence not be repaired in a directed fashion. This crosshatched. The lengths of the double-strand gap, the half proposal shares some features with that made by Alani gaps, and the regions of symmetric strand exchange can be et al. (1994), and elaborated upon by Schwacha and variable. (D and E) Resolution without crossing over by cleav- Kleckner (1995), for how the distribution of nicks asso- age of Holliday junctions results in both the cut chromosome ciated with the resolution of meiotic recombination in- and the repair template chromosome receiving newly synthesized DNA (and, hence, the potential for errors). Note the termediates could bias the repair of heteroduplex DNA potential positioning of strand discontinuities. and help explain the paucity of certain classes of recombinants. In the experiments presented here, we test the hypothesis that mismatch correction is responsible for one chromosome had the recognition site for the endo- restoration of mismatches on the template chromosome nuclease (HO) that made the DSB (Figure 1A), the by monitoring the reversion of a marker near the site chromosome that had the DSB can be identified in cases of a DSB in strains that are defective in mismatch repair where exchange of outside markers does not occur. The because of mutations in the PMS1, MLH1 or MSH2 genes. DSB repair model, as detailed by Szostak et al. (1983), predicts that newly synthesized DNA, and hence new errors, are inherited on both the originally broken chro- MATERIALS AND METHODS mosome and the chromosome that served as the tem- Yeast strains: The yeast strains used are listed in Table 1. plate (Figure 1). In contrast, we found that the errors The basic TRP1-HIS3 module and the positions of the alleles are inherited almost exclusively by the chromosome that are given in Figure 1 and described in McGill et al. (1990, originally had the DSB. We entertained two classes of 1993) and Strathern et al. (1995). The pms1::LEU2 disrup- Misincorporations During DSB Repair 1527

Figure 2.—Resolution models that restrict revertants to the repaired chromosome. (A) Asymmetric gap repair followed by resolution of the two Holliday junctions with the sense confining the new synthesis to the repaired chromosome. (B) Resolution of a symmetric double Holliday junction intermediate by topoisomerase. (C) Traveling replication bubble yielding conservative DNA replication. tion allele was made by single-step gene disruption using a either in liquid or as patches started from single colonies. The plasmid (pWJ401) provided by Dr. Rodney Rothstein. The galactose-induced cultures included some revertants that were mlh1-267::LEU2 allele was made by transformation with a frag- spontaneous in origin. These could be identified as strains ment of pJS267, which has the LEU2 gene as a substitution that were heterozygous for the his3 alleles and that retained for codons 30–702 of MLH1 flanked by 679 bases 5Ј and 476 the HO site and, hence, the ability to be galactose induced to bases 3Ј of the deleted MLH1 region. The msh2-265::LEU2 HIS3. allele was made by transformation with a fragment of pJS265 Media: The media in these experiments were prepared as that has the LEU2 gene as a substitution for codons 11–768 described in Sherman et al. (1986) and McGill et al. (1990). of MSH2 flanked by 608 bases 5Ј and 536 bases 3Ј of the Galactose induction was performed by shifting cells from syn- deleted MSH2 region. thetic complete medium minus uracil plus 5% raffinose (SC- Genetic analysis: Coupling of the TRP1 revertant alleles to Uraϩraffinose) to SC-Ura plus 2% galactose overnight. Ali- MAT and CRY1 was established by classical genetics, as de- quots were titered and plated to detect Hisϩ recombinants scribed in McGill et al. (1990). In brief, patches of the a/␣ and Trpϩ revertants. Frequencies were determined from the diploid revertants were sporulated. Coupling of the cry1 and median value of 11 independent cultures. MAT alleles was determined by replica plating the sporulated cultures onto YEPD plates containing 1 mg/ml cryptopleurine. The mating type of the resulting patch of cryptopleurine- RESULTS resistant spores was then determined by the ability to mate to MATa and MAT␣ strains (DC14 and DC17). The coupling of Reversion of mutant trp1 alleles near the site of a DSB the revertant TRP1 allele to MAT was determined from the repair event is elevated only on the chromosome that sporulated cultures by the ability to mate to and complement had experienced the DSB (Strathern et al. 1995). We the TrpϪ defect of MATa and MAT␣ trp1 tester strains (GRY7 70 and GRY772). In the fluctuation tests, the extent can induce a site- and chromosome-specific DSB in of induction of the HO endonuclease–mediated DSB was de- these diploid strains because there is a recognition site termined by testing several hundred Uraϩ colonies for changes for the HO endonuclease between the trp1 and his3 in the his3 and MAT alleles and for loss of the HO site. Specifi- genes on the cry1 MATa-inc chromosome. The MATa- cally, the diploids were tested for mating phenotype to deter- inc and MAT␣-inc alleles carry defective HO recognition mine whether they retained the a/␣ mating phenotype that is indicative of heterozygosity of MATa and MAT␣, and they sites, thus, the site between trp1 and his3 is the only were tested for heterozygosity of the his3 alleles by scoring available target for HO. The HO gene is under the their ability to give rise to UV-induced Hisϩ recombinants. control of a galactose-regulated promoter (Jensen and Those diploids that were heterozygous for the his3 alleles and Herskowitz 1984). If the observed bias favoring the carried the pGALHO plasmid were tested for the presence of inheritance of the reverted TRP1 allele by the cut chro- the HO cleavage site by monitoring the ability of growth on galactose to promote the formation of Hisϩ recombinants. The mosome reflects directed mismatch repair of errors revertants in the experiment that were used to demonstrate made on the template chromosome, then we predict the chromosome bias came from independent cultures grown that removal of the mismatch repair system should allow 1528 C. B. McGill, S. L. Holbeck and J. N. Strathern

TABLE 1 Yeast strains

Strain Genotype Source Haploids GRY1079 MATa-inc::[trp1-488 HO-site his3-192] cry1 leu2-⌬1 ade2-101 lys2-801 McGill et al. (1993) trp1-⌬1 his3-⌬200 ura3-52 GRY1640 MATa-inc::[trp1-488 HO-site his3-192] cry1 leu2-⌬1 ade2-101 lys2-801 This study trp1-⌬1 his3-⌬200 ura3-52 pms1::LEU2 GRY1733 MATa-inc::[trp1-488 HO-site his3-192] cry1 leu2-⌬1 ade2-101 lys2-801 This study trp1-⌬1 his3-⌬200 ura3-52 mlh1-267::LEU2 GRY1735 MATa-inc::[trp1-488 HO-site his3-192] cry1 leu2-⌬1 ade2-101 lys2-801 This study trp1-⌬1 his3-⌬200 ura3-52 msh2-265::LEU2 GRY1197 MAT␣-inc::[trp1-488 his3-621] leu2-⌬1 tyr7-1 trp1-⌬1his3-⌬200 ura3-52 Strathern et al. (1995) GRY1642 MAT␣-inc::[trp1-488 his3-621] leu2-⌬1 tyr7-1 trp1-⌬1 his3-⌬200 ura3-52 This study pms1::LEU2 GRY1734 MAT␣-inc::[trp1-488 his3-621] leu2-⌬1 tyr7-1 trp1-⌬1 his3-⌬200 ura3-52 This study mlh1-267::LEU2 GRY1736 MAT␣-inc::[trp1-488 his3-621] leu2-⌬1 tyr7-1 trp1-⌬1 his3-⌬200 ura3-52 This study msh2-265::LEU2 GRY770 MATa arg4-17 lys5 trp1-⌬1 McGill et al. (1990) GRY772 MAT␣ arg4-17 lys5 trp1-⌬1 McGill et al. (1990) DC14 MATa his1 Cold Spring Harbor Laboratory DC17 MAT␣ his1 Cold Spring Harbor Laboratory Diploids GRY1198 GRY1079 X GRY1197 [pGALHO] MATa-inc::[trp1-488 HO-site Strathern et al. (1995) his3-192] cry1/MAT␣-inc::[trp1-488 his3-621] GRY1657 GRY1640 X GRY1642 [pGALHO] MATa-inc::[trp1-488 HO-site This study his3-192] cry1 pms1::LEU2/MATa-inc::[trp1-488 his3-621] pms1::LEU2 GRY1731 GRY1733 X GRY1734 [pGALHO] MATa-inc::[trp1-488 HO-site This study his3-192] cry1 mlh1-267::LEU2/MAT␣-inc::[trp1-488 his3-621] mlh1-267::LEU2 GRY1732 GRY1735 X GRY1736 [pGALHO] MATa-inc::[trp1-488 HO-site This study his3-192] cry1 msh2-265::LEU2/MATa-inc::[trp1-488 his 3-621] msh2-265::LEU2 recovery of TRP1 revertants made during DSB repair reversion was accompanied by an exchange of the out- on the template chromosome. side markers CRY1 and MAT (McGill et al. 1990). For Reversion of trp1-488 in a pms1 background: We tested noncrossover diploids, the chromosome that had the this hypothesis first in diploids disrupted for PMS1 DSB was identified by the cry1 and MATa-inc alleles, (Kramer et al. 1989), one of the yeast homologs of while the template chromosome was CRY1 MAT␣-inc. the E. coli mutL gene. The mutL protein appears to Nearly all the spontaneous revertants occurred without be involved in defining the strandedness of mismatch an associated crossover, and the revertant TRP1 allele repair by recognizing nicks made by mutH in the un- was found equally often on the two chromosomes (Table methylated strand of newly replicated DNA (Modrich 3). In the HO-stimulated revertants, 23% of the events and Lahue 1996). We monitored reversion of the trp1- occurred without crossover. When HO endonuclease 488 allele in a diploid (GRY1657) homozygous for the was expressed, the TRP1 allele was preferentially found pms1::LEU2 disruption, and we observed a 10-fold on the cut chromosome (cry1 MATa-inc) in the pms1- higher spontaneous reversion frequency consistent with defective strain similar to the wild-type diploid. Thus, the expectations of a mutator phenotype for pms1 mu- the chromosome bias in the recovery of the revertant tants (Table 2). When this strain was grown on galactose allele was not PMS1 dependent. to induce HO endonuclease and initiate DSB repair, we Reversion of trp1-488 in a mlh1 background: The observed a further increase in the reversion frequency MLH1 gene is another yeast homolog of the mutL gene of the trp1-488 allele, but not to a level demonstrably of E. coli. The diploid strain GRY1731, which is homozy- different than what was observed for the Pmsϩ strain. gous for the mlh1::LEU2 disruption, showed a spontane- The pms1 mutation did not alter the observed efficiency ous reversion frequency for trp1-488 sixfold higher than of induction of the DSB or the induction of HIS3 recom- wild-type, consistent with the expectations of a mutator binants on the other side of the DSB. phenotype for mlh1 mutants (Table 2). When HO endo- Meiotic analysis was used to determine which chromo- nuclease was induced in the mlh1 diploid, we observed some carried the reverted TRP1 allele and whether the a further increase in the frequency of Trpϩ revertants Misincorporations During DSB Repair 1529

TABLE 2 Reversion frequency

Before induction After induction Reversion of trp1-488 (rafﬁnose) (galactose) Correcteda Wild-type [GRY1198 (pGALHO)] (10% HO induction) Trpϩ 0.3 ϫ 10Ϫ8 11 ϫ 10Ϫ8 107 ϫ 10Ϫ8 Hisϩ (recombinants) 0.4 ϫ 10Ϫ4 68 ϫ 10Ϫ4 680 ϫ 10Ϫ4 pms1 [GRY1657 (pGALHO)] (13% HO induction) Trpϩ 3.1 ϫ 10Ϫ8 16 ϫ 10Ϫ8 99 ϫ 10Ϫ8 Hisϩ (recombinants) 0.2 ϫ 10Ϫ4 99 ϫ 10Ϫ4 760 ϫ 10Ϫ4 mlh1 [GRY1731 (pGALHO)] (16% HO induction) Trpϩ 2.0 ϫ 10Ϫ8 19.7 ϫ 10Ϫ8 106 ϫ 10Ϫ8 Hisϩ (recombinants) 0.9 ϫ 10Ϫ4 80 ϫ 10Ϫ4 500 ϫ 10Ϫ4 msh2 [GRY1732 (pGALHO)] (5% HO induction) Trpϩ 2.0 ϫ 10Ϫ8 7.6 ϫ 10Ϫ8 112 ϫ 10Ϫ8 Hisϩ (recombinants) 0.1 ϫ 10Ϫ4 35 ϫ 10Ϫ4 700 ϫ 10Ϫ4 Frequencies represent median value for 11 independent cultures. a Corrected ϭ (induced Ϫ spontaneous)/level of induction. to a level similar to the induced level in the wild-type (GRY1732) defective in the MSH2 gene. The msh2 dip- strain. The mlh1 mutation did not affect the induction loid exhibited a spontaneous mutator phenotype, but of HIS3 recombinants. As observed for pms1, there was among cells with an HO-induced DSB, the reversion no chromosome bias for spontaneous revertants, but frequency was similar to that observed in wild-type cells we observed a strong bias in DSB-associated revertants and the pms1 and mlh1 strains described above (Table favoring the recovery of the revertant allele on the cut 2). The spontaneous revertants isolated from msh2 cul- chromosome (cry1 MATa-inc), as deﬁned in the 19% of tures were randomly distributed between the two chro- revertants not associated with a crossover. Thus, MLH1 mosomes (Table 3). In contrast, the DSB-associated re- is not required for the asymmetry in the genetic cou- vertants showed the same biased distribution, favoring pling of the revertant allele. thecutchromosomeoverthetemplatechromosomeseen Reversion of trp1-488 in a msh2 background: We moni- in wild-type or the pms1 and mlh1 mutant backgrounds. tored the reversion of the trp1-488 allele in a diploid

DISCUSSION TABLE 3 In addition to its role in genome duplication, DNA Distribution of the revertant allele synthesis is a crucial part of some mechanisms of DNA between the two chromosomes damage repair and recombination. The net stability of the sequence of the genome depends on the ﬁdelity of Chromosome with the each of these processes. The elevated mutation rates in revertant allele cells that are defective in mismatch correction demon- trp1-488 revertants CRY1 MAT␣-inc CRY1 MATa-inc strate the importance of this process in mutation avoidance (reviewed in Modrich and Lahue 1996). How- Wild-type [GRY1198]a Spontaneous 19 15 ever, the relative importance of mismatch correction in HO inducedb 11 94 the various classes of DNA synthesis remains unclear. pms1 [GRY1657] Understanding the origin of spontaneous mutations will Spontaneous 17 13 require the determination of what DNA polymerases HO inducedb 15 93 and accessory factors contribute to these multiple DNA mlh1 [GRY1731] synthesis processes. In these experiments, we monitored Spontaneous 26 26 the ﬁdelity of DNA synthesis associated with DSB repair. HO inducedb 137 msh2 [GRY1732] Previous experiments demonstrated that there is an ele- Spontaneous 13 19 vated mutation rate in the region (0.3 kb) of DSB repair HO inducedb 262events, and that those mutations are recovered far more

a Strathern often on the repaired chromosome than on the tem- Includes data from et al. (1995). Strathern b Spontaneous events among the galactose-induced re- plate chromosome ( et al. 1995). In this revertants were identiﬁed and discarded as described in materi- port, we focused on the chromosome bias in the recov- als and methods. ery of misincorporations associated with DSB repair. 1530 C. B. McGill, S. L. Holbeck and J. N. Strathern

Just as the distribution of gene convertants and post- junctions. The use of those strand discontinuities to meiotic segregants and their association with meiotic direct mismatch repair would result in fixation of the recombination constrains models to explain their ori- new sequence on the repaired chromosome. Schwacha gins (Holliday 1964; Meselson and Radding 1975), and Kleckner (1995) proposed a similar model for how the asymmetric inheritance of mutations associated with the nicks left from the resolution of Holliday junctions DSB repair constrains models for how they are gener- could account for directed mismatch repair and the ated. For the purposes of this discussion, the excess paucity of Ab5:3 tetrads. We also noted that the strand of 5:3 tetrads over Ab5:3 tetrads in yeast provides an discontinuities at the end of the gap repair synthesis importantparallel (Fogel et al. 1981; Radding 1982). In might also still be present to guide mismatch correction. 5:3 tetrads, the sectored colony carries the chromosome On the template chromosome, the strand break at the defined as the recipient, whereas in Ab5:3 tetrads, the end of synthesis would be on the same strand as the sectored colony carries the chromosome defined as the strand breaks, resulting from resolution of the Holliday donor. This distinction can only be made for tetrads junctions. Nick-directed mismatch repair would then that do not have a crossover for outside markers. As promote restoration. On the repaired chromosome, the discussed below, the mechanisms that constrain against strand break at the end of the synthesis would be on Ab5:3 in meiosis can also function as mechanisms that the opposite strand from the strand break from the determine the chromosome that inherits errors associ- cleavage of the Holliday junctions. The presence of ated with mitotic DSB repair. breaks on both strands could lead to randomized mis- The recombination events studied here are initiated match correction. by site-specific, double-strand cleavage. The symmetric Our results are not consistent with the model that form of the DSB repair model for genetic recombina- the chromosome bias in recovering revertants associ- tion as described by Szostak et al. (1983) allows the ated with DSB repair is the result of nick-directed mis- generation of 6:2 gene convertant tetrads resulting from match correction. The protein encoded by the mutS either the repair of double-strand gaps or by the mis- homolog MSH2 (Reenan and Kolodner 1992) is di- match correction of heteroduplexes in regions of single- rectly involved in mismatch recognition (Alani et al. or symmetric-strand exchange. Repair of a double- 1995; Alani 1996) and, hence, is a prime candidate for strand gap requires that both strands of DNA spanning an essential function in any model invoking directed the gap be resynthesized, while repair of a single-strand mismatch repair. The proteins encoded by the mutL gap requires only one strand of DNA to be synthesized. homologs MLH1 and PMS1 form a heterodimer, and In the symmetric form of the DSB repair model, newly they are both required for mismatch repair (Prolla et synthesized DNA is inherited by both the repaired and al. 1994a,b). We tested both pms1 and mlh1 strains be- the template chromosome (Figure 1). In the experi- cause it is possible that they provide different functions ments reported here, we tested the hypothesis that the and could act independently in correcting mistakes failure to recover revertants on the template chromo- made during DNA synthesis associated with DSB repair. some results from directed mismatch repair of base sub- We found, however, that defects in PMS1, MLH1 or stitutions leading to restoration on the template chro- MSH2 do not change the bias favoring recovery of the mosome. We can define the template chromosome in revertants on the repaired rather than the template repair events that are not associated with crossing over chromosome. Furthermore, the frequency of revertants because the recognition site for HO endonuclease is associated with DSB repair is the same in the mismatch present on only one of the two chromosomes. This is repair-defective strains as it is in the wild-type back- in contrast to most meiotic experiments, where the two ground. We do not see the twofold increase in mutation alleles have similar probabilities of acting as initiator frequency in the repair-defective strains that was pre- and the donor is inferred from the resulting postmeiotic dicted from the model that mismatches are repaired distribution of markers. randomly in wild-type strains. This suggests that base Resolution of a double Holliday junction joint mole- substitutions made during DSB repair were not sub- cule to produce a noncrossover involves cleavage of the strates for the products of these genes, even when the same strand of each duplex at both Holliday junctions misincorporation was on the repaired chromosome. It (Szostak et al. 1983; Alani et al. 1994). On the template remains possible that there are mismatch detection chromosome, the newly synthesized DNA (and hence mechanisms independent of PMS1, MLH1, and MSH2 any errors) will always be on the strand that was cut to that are dedicated to the DSB repair process. Such pro- resolve the Holliday junctions (Figure 1, D and E). The teins could be specifically associated with the polymer- use of those strand discontinuities to direct correction ases involved in DSB repair and/or those proteins spe- of misincorporations made during DSB repair would cifically involved in the formation or resolution of the result in restoration of the template chromosome se- recombination intermediate. quence (Strathern et al. 1995). On the repaired chro- The elevated reversion of the trp1-488 nonsense allele mosome, the newly synthesized DNA will always be on near the site of a DSB repair is almost completely depen- the strand that was not cleaved to resolve the Holliday dent on REV3, suggesting that the translesion polymer- Misincorporations During DSB Repair 1531 ase (Pol ␨) encoded by REV3 (Nelson et al. 1996) is chromosome received the strand with the newly synthe- recruited to recombination intermediates and makes sized DNA. misincorporation errors on these presumably undam- A second way to resolve a symmetric double Holliday aged templates (Holbeck and Strathern 1997). Thus, junction as a noncrossover without leaving heteroduplexes the revertants of trp1-488 studied here could be the on both chromosomes is to use topoisomerases rather result of a polymerization step that is not reflective of than cleavage of the Holliday junctions (Nasmyth 1982; the majority of DSB repair syntheses. That is, they might Thaler et al. 1987; Hastings 1988; McGill et al. 1989). represent events in which the translesion polymerase is Resolution of a double Holliday junction by topoisomer- recruited because the template is not used efficiently ases results in all the newly synthesized DNA being recov- by the standard polymerases. For that reason, they might ered on the repaired chromosome (Figure 2B). Support reflect a pathway that does not have associated mismatch for this mechanism of resolution has been reported, repair functions required for replication fidelity. In its based on the strandedness of heteroduplexes on oppo- role as a translesion polymerase, it might be counterpro- site sites of an initiation site in meiosis (Gilbertson ductive for Pol ␨ to be coupled to a mismatch detection and Stahl 1996) and in homothallic switching (McGill system that is biased toward removing the newly synthe- et al. 1989). sized strand. One would still be left with the puzzle of DSB repair can be accomplished without both sides of why lesions made by Pol ␨ are preferentially inherited the break invading the template chromosome (Resnick by the repaired chromosome. 1976). In this class of models, DNA synthesis proceeds Tran et al. (1996) found that the ability to repair on the template chromosome from only one side until single base deletions is dependent on how they are the region of the DSB has been spanned (Figure 2C). made. They observed that the rate of single base Interaction of the newly synthesized DNA and the other frameshift reversions (deletions) are elevated in a rad52 side of the broken chromosome can allow repair of background, but this mutator phenotype of rad52 is the DSB without the formation of a double Holliday not made more dramatic by an additional defect in junction intermediate. This pathway does not produce mismatch repair (pms1). They conclude that the dele- reciprocal crossovers, but it can yield the noncrossover tions are not made during semiconservative DNA syn- events that are the focus of this study. thesis, but instead, that they occur during an error- The results presented here demonstrate that not all prone process that does not yield a substrate that can DNA synthesis is equivalent with regard to mutation be detected by mismatch repair proteins. Roche et al. avoidance mechanisms. This was first demonstrated by (1995) demonstrated that the mutator phenotype caused Santos and Drake (1994) in experiments showing that by rad52 is dependent on REV3 (Nelson et al. 1996). the net misincorporation rate for T4 replication is Combined, these results also suggest that mutations higher than for its host, E. coli, and is not subject to made by Pol ␨ are not substrates for mismatch repair. mismatch correction. Starvation in E. coli leads to ele- Three other classes of models have been proposed for vated mutation rates (reviewed by Foster 1993), which how the double Holliday junction might be a common may reflect decreased mismatch repair capacity (Harris intermediate in meiotic recombination and not yield et al. 1997), suggesting that cells may be able to adjust Ab5:3 tetrads. Szostak et al. (1983) recognized that the the fidelity of DNA synthesis in response to the environ- symmetric intermediate predicted the common forma- ment. Localized elevated mutation rates have been im- tion of Ab5:3 tetrads, and they proposed constraints on plicated in the origin of the somatic diversity of immu- the formation of regions of strand exchange and the noglobin genes (Neuberger and Milstein 1995), but resolution of the Holliday junctions that would prohibit it remains to be determined whether that process is those classes of tetrads. Specifically, they suggested that subject to mismatch correction. the extent of asymmetric strand exchange was much Our results highlight the complexity of the issue of greater on one side of the “bubble,” and that resolution the origin of spontaneous mutations. Mismatch correc- of the Holliday junctions was controlled so that the tion is a critical component of the processes that define exchanged strand was inherited by the chromosome the fidelity of genome replication. Defects in MSH2, that had the initiating DSB. Note that in this view, the PMS1 or MLH1 cause substantial increases in the levels template chromosome gets most of the new DNA synthe- of spontaneous mutagenesis in yeast. These functions, sis. A related model can be used to explain the distribu- however, appear to play little role in defining the mutation of revertants associated with DSB repair (Figure tion frequency associated with DSB repair. Further- 2A). In this view, the region of double-strand gap would more, while these functions have been reported to alter have to be small relative to the single-strand gap, and the mitotic recombination (Datta et al. 1997; Negritto et single-strand recision (and hence required resynthesis) al. 1997), as well as the pattern of inheritance of gene would have to be primarily in one direction from the conversions and PMS events in meiosis (Borts et al. DSB. In contrast to the model for meiotic recombina- 1990; Hunter and Borts 1997), they did not alter the tion, the resolution of the Holliday junctions in mitotic chromosome bias in the recovery of revertants of trp1- DSB repair would have to be biased so that the repaired 488 stimulated by DSB repair. It remains to be deter- 1532 C. B. McGill, S. L. Holbeck and J. N. Strathern mined whether that bias represents the use of alternate from Saccharomyces cerevisiae: homology to procaryotic MutL and HexB. J. 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