Sequence Divergence Impedes Crossover More Than Noncrossover Events During Mitotic Gap Repair in Yeast

Caroline Welz-Voegele and Sue Jinks-Robertson1 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710 Manuscript received April 11, 2008 Accepted for publication May 8, 2008

ABSTRACT Homologous recombination between dispersed repeated sequences is important in shaping eukaryotic genome structure, and such ectopic interactions are affected by repeat size and sequence identity. A transformation-based, gap-repair assay was used to examine the effect of 2% sequence divergence on the efﬁciency of mitotic double-strand break repair templated by chromosomal sequences in yeast. Because the repaired plasmid could either remain autonomous or integrate into the genome, the effect of sequence divergence on the crossover–noncrossover (CO–NCO) outcome was also examined. Finally, proteins important for regulating the CO–NCO outcome and for enforcing identity requirements during recombination were examined by transforming appropriate mutant strains. Results demonstrate that the basic CO–NCO outcome is regulated by the Rad1-Rad10 endonuclease and the Sgs1 and Srs2 helicases, that sequence divergence impedes CO to a much greater extent than NCO events, that an intact mismatch repair system is required for the discriminating identical and nonidentical repair templates, and that the Sgs1 and Srs2 helicases play additional, antirecombination roles when the interacting sequences are not identical.

ECOMBINATION is a high-ﬁdelity process that persed throughout the genome. Such ectopic inter- R copies information from one DNA duplex to reactions are important for shaping genome structure, pair single- or double-strand discontinuities in another with NCO events likely driving the concerted evolution DNA duplex (reviewed by Krogh and Symington of multigene families and COs leading to various types 2004). Repair events involve either the unidirectional of genome rearrangements. In mitotic studies using transfer of information between duplexes or the recip- model recombination substrates in the yeast Saccharo- rocal exchange of information, which will be referred myces cerevisiae, the rate of ectopic interactions is directly to here as noncrossover (NCO) and crossover (CO) proportional to repeat size (Ahn et al. 1988; Hayden events, respectively. In classic models of homologous and Byers 1992; Jinks-Robertson et al. 1993; Inbar recombination, NCO and CO events derive from et al. 2000) and inversely proportional to the level of alternative cleavage of a common intermediate known sequence divergence (Datta et al. 1997; Chen and as a Holliday junction (HJ), which corresponds to the Jinks-Robertson 1998). point where the single strands of the interacting The major barrier to recombination between di- duplexes switch pairing partners. In more recent re- verged sequences in yeast derives from antirecombina- combination models, however, some NCO events pro- tion activity of the mismatch repair (MMR) system ceed through an intermediate that cannot be processed (reviewed in Surtees et al. 2004), which is best known into a CO. The template for recombinational repair is for its roles in removing DNA replication errors and typically a homologous chromosome or sister chroma- repairing mismatches in meiotic recombination inter- tid, but because recombination is a homology-driven mediates (reviewed in Harfe and Jinks-Robertson process, it also can engage repetitive sequences dis- 2000; Kunkel and Erie 2005). There are three major yeast complexes involved in mismatch removal and in antirecombination: MutSa, MutSb, and MutLa com- This article is dedicated to the memory of Caroline Welz-Voegele, who posed of Msh2-Msh6, Msh2-Msh3, and Mlh1-Pms1, died on September 12, 2007. During her 10 years with the Jinks-Robertson respectively. MutSa and MutSb bind directly to mis- group, Caroline made numerous experimental and intellectual contribu- a a b tions, she was generous with her time and knowledge, and she served as a matches, while MutL couples MutS / -mediated mis- mentor and role model for all who passed through the lab. Caroline is match recognition to the appropriate downstream greatly missed and fondly remembered by her friends and colleagues at processing steps, the precise mechanism(s) of which Emory University and Duke University. remain obscure. Additional proteins implicated in 1Corresponding author: Department of Molecular Genetics and Microbi- ology, Duke University Medical Center 3020, 228 Jones Bldg., Research the repair of mismatches include the Rad1-Rad10 Dr., Durham, NC 27710. E-mail: [email protected] endonuclease (Kirkpatrick and Petes 1997), the

Genetics 179: 1251–1262 ( July 2008) 1252 C. Welz-Voegele and S. Jinks-Robertson

Exo1 exonuclease (Tishkoff et al. 1997), and the derivative containing a 1.7-kb BamHI genomic HIS3 fragment. PCNA sliding clamp ( Johnson et al. 1996; Umar et al. Following treatment with T4 DNA polymerase and T4 kinase, the fragment was inserted at the SmaI site of the LEU2-CEN 1996). Rad1-Rad10 and Exo1 are also important in ikorski ieter icholson vector pRS315 (S and H 1989). The resulting antirecombination (N et al. 2000), but plasmids pSR515 and pSR516 contain the HIS3 gene in the PCNA plays little, if any, role in this process (Stone opposite and same orientation, respectively, as the vector lacZ et al. 2008). In contrast, the helicase Sgs1 is important gene. The HIS3 gene of these plasmids contains ,10 bp of in antirecombination (Myung et al. 2001; Spell and identity to the his3D200 allele at the endogenous HIS3 locus. Jinks-Robertson 2004), but has no known role in The HIS3-18 allele of pSR612 contains 18 silent, randomly distributed mutations (see supplemental Figure 1) and is 98% the repair of mismatches. Thus, although there are identical to the standard HIS3 gene. The mutations were similarities in the MMR-directed editing of replication introduced by subjecting pSR515 to sequential rounds of site- and recombination intermediates, there are genetic directed mutagenesis using the Chameleon double-stranded, and presumably mechanistic differences as well. site-directed mutagenesis kit (Stratagene). All mutations were In yeast, chromosomal sequences can serve as a tem- confirmed by sequencing, and pSR612 was able to fully complement the His phenotype of a his3D200 strain. plate for the faithful repair of a linear, gap-containing pSR800 contains a 39-truncated, but otherwise wild-type plasmid and such gap-repair reactions were instrumen- (WT), his3 allele inserted into the CAN1 coding sequence. tal in the development of the double-strand break Because this allele contains no polymorphisms, it is referred to (DSB) repair model of recombination (Szostak et al. as his3-0,D39 to distinguish it from the similarly truncated allele 1983). Of particular significance was the observation that contains the engineered silent changes (his3-18,D39). The can1This3D39 allele was constructed as follows. First, the that the repaired plasmid either remains autonomous smallest KpnI fragment of pSR515 was deleted, thereby or integrates into the host genome (Orr-Weaver and truncating HIS3 at an internal KpnI site and eliminating the Szostak 1983), outcomes presumed to reflect cleavage last 11 amino acids of the encoded protein. An 834-bp BamHI- of a Holliday junction intermediate to generate NCO or KpnI fragment from the resulting plasmid (pSR798) was then CO products, respectively. Subsequent studies have treated with T4 DNA polymerase and inserted at the MscI site of pSR797. pSR797 is a pUC9 derivative containing an 1122-bp confirmed that plasmid-based repair assays generally CAN1 fragment (121 to 11141 of the 1773 nt CAN1 ORF) recapitulate the genetic requirements and features of inserted at the SmaI site of the vector polylinker. The resulting DSB-initiated chromosomal recombination (Bartsch plasmid pSR800 contains the his3-0,D39 allele in an orientation et al. 2000) and hence are a useful model for studying opposite to that of the CAN1 sequences. pSR801 contains the T basic recombination processes. In the experiments can1 his3-18,D39 allele and was constructed in the same manner as pSR800, but starting from pSR612. reported here, a gap-repair assay was used to examine pSR840 contains the his3DBgl allele and was used as the the regulation of mitotic recombination fidelity, with template for producing the linear ‘‘gapped vector’’ PCR an emphasis on how sequence divergence affects the fragment for transformation assays. pSR840 was derived by CO–NCO decision in different genetic backgrounds. first inserting a SacI-SalI fragment containing the full-length HIS3 gene (from pSR516) into SacI/SalI-digested pRS306, an These studies support roles for the Srs2 and Sgs1 ikorski ieter helicases, the Rad1-Rad10 endonuclease, and MutSb integrating URA3 vector (S and H 1989). The internal 60-bp BglII fragment was then deleted to generate the in regulating the CO–NCO outcome, confirm that the his3DBgl allele. Finally, a Klenow-treated HinfI-EcoO109 frag- regulation of recombination fidelity depends on activity ment containing the ARS4 replication origin of pRS315 of the MMR machinery, and demonstrate that sequence (Sikorski and Hieter 1989) was ligated to AatII-digested divergence impedes CO events to a much greater extent plasmid that had been treated with T4 DNA polymerase. than NCO events. Yeast strains: Transformation with PstI/PvuII-digested pSR800 or pSR801 was used to replace the CAN1 allele in T SJR328 (MATa ade2-101oc his3D200 ura3-Nhe lys2DRV hisG leu2-R Gal1;Chen and Jinks-Robertson 1999) with the T T MATERIALS AND METHODS can1 his3-0,D39 or can1 his3-18,D39 allele, respectively. Fol- lowing selection of transformants on SC arg 1CAN, re- Media and growth conditions: Yeast strains were grown placements were confirmed by PCR. SJR1500 contains the nonselectively in YEP (1% Bacto-yeast extract, 2% Bacto can1This3-0,D39 allele, while SJR1501 contains the can1T peptone), supplemented with 2% glucose and 500 mg/ml his3-18,D39 allele. adenine hemisulfate (YEPD). For selection of strains contain- Repair-defective derivatives of SJR1500 and SJR1501 were ing the kan or hph marker, YEPD was supplemented to 200 constructed by targeted gene deletion. The MSH2 gene was mg/ml with geneticin or to 300 mg/ml with hygromycin B, deleted by one-step gene replacement using AatII/XbaI- respectively. For selective growth, synthetic complete (SC) digested pDmsh2 (msh2DThisG-URA3-hisG plasmid; Earley (Sherman 1991) medium contained 2% glucose and all but and Crouse 1998). Following the selection of Ura1 trans- the one relevant amino acid or base (e.g.,SChis for the formants, YEPD-purified colonies were patched to 5-FOA selection of His1 recombinants). Canavanine-resistant colo- medium to select for loss of URA3 and one copy of hisG. nies were selected on SC arg medium supplemented with SJR1476 and SJR1477 are the resulting msh2DThisG derivatives 60 mg/ml l-canavanine. Ura segregants were selected on of SJR1500 and SJR1501, respectively. msh6DThisG (SJR2047 SC plates supplemented with 0.1% 5-flouroorotic acid (US and SJR2048), msh3DThisG (SJR2054 and SJR2055), or rad1- Biological). All growth was at 30°. ThisG derivatives (SJR2111 and SJR2112) of SJR1500 and Plasmids: A 938-bp fragment containing the 663 bp HIS3 SJR1501 were similarly constructed using SacI/EcoRI-digested ORF together with 199 bp of upstream and 76 bp of down- pBUH-msh6ThisG-URA3 (Kramer et al. 1996), EcoRI-digested stream sequence was PCR amplified from pSR22, a pUC7 pEN33 (Datta et al. 1996) or SalI/EcoRI-digested pR1.6 Sequence Divergence and Recombination 1253

(Higgins et al. 1983), respectively. The pms1D derivatives of SJR1500 and SJR1501 (SJR2147 and SJR2148, respectively) were constructed by standard two-step allele replacement using BstXI-digested pJH523 (Kramer et al. 1989). The mlh1DTkanMX4 (SJR2156 and SJR2157), sgs1DTkanMX4 (SJR2122 and SJR2163), and srs2DTkanMX4 (SJR2123 and SJR2160) derivatives of SJR1500 and SJR1501 were constructed by transformation with PCR deletion cassettes generated using pFA6-kanMX4 (Wach et al. 1994) as a template. Presence of the relevant mutant allele and absence of the corresponding WT allele were confirmed by PCR. Gap-repair experiments: The fragment used for gap-repair assays was produced by PCR amplification of pSR840. Primers HisBglIIF (59-CTCTTGCGAGATGATCCCGC) and HisBglIIR (59-ACCACCGCTCTGGAAAGTGCC), which anneal directly adjacent to the single BglII site that marks the 60-bp deletion, were used to amplify a 5.7-kb fragment with Taq Plus Precision polymerase (Stratagene). After the PCR reaction, the template pSR840 DNA was destroyed using the methylation-sensitive enzyme DpnI. To correct for variation in transformation Figure 1.—The gap-repair assay. The lengths of homology efficiency, the PCR product was mixed with uncut control that flank the gap on the 59 and 39 sides are 600 bp and ikorski ieter plasmid (pRS315, a CEN-LEU2 vector; S and H 160 bp, respectively. See text for further explanation of the 1989) in a 20:1 weight ratio. system. A high-efficiency transformation protocol was used (Gietz and Woods 2002), with the following modifications. To minimize the culture-to-culture variation seen with some of the gene products involved in this regulation. Because the repair-defective strains, five colonies were pooled to our earlier studies demonstrated that a single potential inoculate 5 ml of liquid YEPD. After overnight growth, 1 ml was transferred to a prewarmed flask containing 50 ml YEPD, mismatch is sufficient to trigger the mitotic antirecom- and the flask was incubated on a rotary shaker at 200 rpm for bination activity of the yeast MMR system (Datta et al. 3 hr. Cells were harvested by centrifugation at room temper- 1997), it was essential to use 100%-identical sequences ature, washed twice, and resuspended in 360 mlofH2O. as a control against which to gauge the effects of se- Twenty-five-microliter samples of the cell suspension were quence divergence. Genetic assays designed to simulta- pelleted by centrifugation, the supernatant was removed, and 120 ml of PEG 3350 (50%, w/v) were layered over the pellets. neously detect both NCO and CO events typically rely Sixty microliters of freshly prepared transformation mix [18 ml on the transfer of WT sequence to correct an auxotro- of 1 m LiAc; 5 ml of 10 mg/ml boiled salmon sperm carrier phic allele, thereby precluding absolute identity be- DNA; 17 mlH2O; 20 ml of a gapped vector/control plasmid tween the interacting sequences. Such identity was 1 mix (40 ng 2 ng)] were added and the tubes were vortexed achieved in the current study by using a transforma- for 1 min. Following incubation at 42° for 1 hr, cells were pelleted, resuspended in 550 ml of sterile water, and vortexed tion-based, gap-repair assay in which the template for for 1 min. One-hundred-microliter aliquots were plated on the gap-filling reaction is a truncated, but otherwise WT, SC his plates to select recombinants and on SC leu plates to allele. Recombination between the two mutant alleles— determine transformation efficiency. For each experiment, extrachromosomal gapped and chromosomal truncated— four replicates of each transformation were performed and produces a selectable, WT allele on the plasmid. If the each experiment was repeated at least once. Colonies were counted after 3 days of growth on the selective media and gap- gapped plasmid is furthermore capable either of auton- repair frequency was calculated as the ratio of His1 trans- omous replication or of integrating into the genome, it formants to Leu1 transformants. is straightforward to distinguish NCO from CO events, The proportions of NCOs and COs among the gap-repair respectively (Orr-Weaver and Szostak 1983). events in each strain were determined by directly patching 50 The HIS3 gene was used as the basis for developing His1 transformants from each of at least 5 independent transformations onto YEPD. Patches were replica plated to 5- the gap-repair assay and was placed on a vector contain- FOA medium and those with full growth after 2 days were ing the URA3 gene and an origin of replication (ARS), scored as NCO events; patches with no growth or only a few but no centromeric (CEN) sequence (Figure 1). This papillae were scored as CO events. Random genomic DNAs plasmid was then used as a PCR template to generate a were analyzed by PCR to confirm the accuracy of the NCO–CO linear (gapped) transformation fragment (see materials assignment. and methods). To obtain an identical (‘‘homologous’’) repair template, a mutant his3 allele missing the C- terminal 11 amino acids (his3-0,D39) was inserted into RESULTS the CAN1 locus of a haploid strain containing a deletion A gap-repair system for assaying homologous and of the endogenous HIS3 coding sequence. To obtain a homeologous recombination: The goal of the current 98%-identical (‘‘homeologous’’) repair template, 18 study was to determine whether sequence divergence silent mutations were engineered into the coding has similar inhibitory effects on mitotic crossover (CO) sequence of the truncated his3 allele (his3-18,D39). and noncrossover (NCO) events, and if so, to identify The his3-0,D39 and his3-18,D39 strains (or appropriate 1254 C. Welz-Voegele and S. Jinks-Robertson

TABLE 1 Gap-repair efﬁciencies using 100- vs. 98%-identical chromosomal donor sequences in different genetic backgrounds

Relevant Substrate His1/Leu1 CO Strain genotype identity (%) frequency proportion CO/NCO SJR1500 WT 100 1.6 6 0.08 0.53 6 0.031 1.1 SJR1501 WT 98 0.66 6 0.035 0.17 6 0.016 0.20 SJR1476 msh2 100 0.83 6 0.045 0.44 6 0.054 0.79 SJR1477 msh2 98 0.57 6 0.085 0.24 6 0.032 0.32 SJR2054 msh3 100 0.97 6 0.052 0.21 6 0.044 0.27 SJR2055 msh3 98 0.53 6 0.031 0.066 6 0.020 0.075 SJR2047 msh6 100 1.7 6 0.11 0.55 6 0.029 1.2 SJR2048 msh6 98 1.6 6 0.089 0.56 6 0.042 1.3 SJR2156 mlh1 100 1.6 6 0.032 0.61 6 0.034 1.6 SJR2157 mlh1 98 1.6 6 0.092 0.54 6 0.059 1.2 SJR2147 pms1 100 1.6 6 0.11 0.55 6 0.037 1.2 SJR2148 pms1 98 1.2 6 0.12 0.42 6 0.019 0.72 SJR2111 rad1 100 1.0 6 0.044 0.052 6 0.018 0.053 SJR2112 rad1 98 0.49 6 0.017 0.020 6 0.024 0.020 SJR2122 sgs1 100 1.2 6 0.065 0.87 6 0.023 6.7 SJR2163 sgs1 98 0.60 6 0.018 0.76 6 0.089 3.2 SJR2123 srs2 100 0.75 6 0.013 0.67 6 0.11 2.0 SJR2160 srs2 98 0.32 6 0.019 0.38 6 0.064 0.61 The mean and standard deviation of the ratio of His1 to Leu1 transformants and of the proportion of CO events obtained in independent transformations is indicated. NCO, noncrossover; CO, crossover.

mutant derivatives) were then transformed in parallel calculated by multiplying the relative gap-repair efficiency using the PCR-generated fragment. Variations in trans- bythepercentageofunstablevs. stable transformants, formation efficiencies in different genetic backgrounds respectively. In the WT strain, the repaired plasmid was (Bartsch et al. 2000; Haghnazari and Heyer 2004) integrated into the yeast genome in about half of the were corrected for by mixing an intact LEU2/ARS/CEN His1 transformants (47% NCOs and 53% COs). plasmid (pRS315; Sikorski and Hieter 1989) with the In terms of their effects on the total efficiency of gap linear fragment prior to transformation. His1 and Leu1 repair between identical substrates, the MMR proteins transformants were selected separately, and the effi- fell into two distinct groups. The efficiency was not ciency of gap repair was calculated as the ratio of His1 to detectably altered in the msh6, pms1,ormlh1 back- Leu1 transformants. The stability of the URA3 marker ground, but there was an approximately twofold de- on the repaired plasmid was then assessed by transferring crease in an msh2 or msh3 mutant. In the case of Msh3, its His1 transformants to medium containing 5-FOA. A loss had no effect on the frequency of NCOs, but stable Ura1 phenotype is diagnostic of a chromosomal reduced CO events fourfold; Msh2 loss was associated URA3 gene and hence plasmid integration at the CAN1 with approximately twofold decreases in both NCO and locus (CO event), whereas an unstable Ura1 phenotype is CO events. The NCO–CO distribution in the msh2 mu- conferred when the URA3 gene is on an autonomous tant was significantly different from that in the msh3 plasmid (NCO event). The primary data obtained mutant (P , 0.001). Given that the only known function following the transformation of WT, msh2, msh3, msh6, for Msh3 is as part of the MutSb complex with Msh2, it mlh1, pms1, rad1, srs2,orsgs1 strains containing either the was surprising that msh2 and msh3 mutants did not his3-0,D39 or his3-18,D39 allele are presented in Table 1. exhibit similar levels of NCO and CO events between Genetic control of homologous gap repair: Compar- identical repeats. It is possible that either Msh2 alone or ison of the gap-repair efficiency in WT vs. mutant strains the Msh2-Msh6 complex, which might be more abun- containing the chromosomal his3-0,D39 allele allows dant in the absence of Msh3 (Drummond et al. 1997), one to ascertain the general effects of the correspond- has subtle effects on recombination. ing gene products on homologous gap repair (HGR). The role of MutSb during gap repair most likely These results are presented in Figure 2, where the reflects an accessory role during the Rad1-Rad10- efficiency of gap repair in each mutant background was dependent processing of branched recombination normalized to that obtained in the WT strain (i.e., the intermediates (see Surtees and Alani 2006). The His1/Leu1 ratio of the WT strain was set to 1.0). The Rad1-Rad10 complex is best known for its essential levels of NCO vs. CO events in each background were function during the incision step of nucleotide excision Sequence Divergence and Recombination 1255

Figure 3.—Homologous and homeologous gap repair (HGR and HeGR, respectively) in a WTstrain. Strains contain- igure F 2.—The genetic control of homologous gap repair. ing a homologous or homeologous repair template (the his3- The total gap-repair efficiency using an identical chromosomal 0,D39 or his3-18,D39 allele, respectively) were transformed template (the his3-0,D39 allele) in each strain background was 1 1 1 with the gapped plasmid and the total (NCO CO) repair measured as the ratio of His to Leu transformants. These ra- efficiency was measured as the ratio of His1 to Leu1 colonies. tios were then normalized to that obtained in the WT strain. These ratios were normalized to that obtained with the ho- The open and shaded areas within each bar correspond to mologous, 100%-identical substrates; the standard deviations the NCO and CO efficiencies, respectively, and were obtained of the total efficiencies are indicated. NCO and CO efficien- by multiplying the total (normalized) efficiency by the propor- cies were obtained by multiplying the total (normalized) effi- tion of the relevant event. The standard deviation of the total ciency by the proportion of the relevant event. Open and efficiencyineachstrainisindicated. shaded bars correspond to HGR and HeGR efficiencies, respectively. repair, where it nicks at the junction of single- and double-stranded DNA (Prakash and Prakash 2000). During recombination, Rad1-Rad10 removes nonho- 2003; Robert et al. 2006). In the gap-repair assay there mologous 39 tails from recombination intermediates was a 2-fold decrease in the overall transformation (Fishman-Lobell and Haber 1992), stimulates dele- efficiency in an srs2 background, and a subtle, 25% tion events between direct repeats (Saparbaev et al. decrease in an sgs1 mutant. While both NCO and CO 1996), facilitates ‘‘ends-in’’ plasmid-chromosome CO events were reduced in the srs2 mutant, there was a events (Schiestl and Prakash 1988; Symington et al. greater reduction in NCO than in CO recombinants (3- 2000), and increases the efficiency of ‘‘ends-out’’ tar- fold and 1.7-fold, respectively), resulting in a 2-fold bias geted gene replacement (Langston and Symington for CO events. In the sgs1 mutant, there was a much 2005). Consistent with previous results, a rad1 mutant more striking shift in the distribution of CO relative to exhibited a specific, 16-fold decrease in COs in our gap- NCO events, with almost 90% of the gap-repaired repair assay, which translated into a 2-fold reduction in plasmids integrating into the genome. This strong CO the total gap-repair efficiency. The requirement of bias can be attributed to a 5-fold reduction in NCOs, Rad1, and by inference the Rad1-Rad10 complex, for some of which may have been converted into CO events. .90% of COs associated with gap repair suggests a key Effects of sequence divergence on gap repair in a role for this protein in the formation of Holliday WT background: The effects of 2% sequence diver- junction-containing intermediates (see discussion). gence on gap repair in a WT background are presented In addition to analyzing the roles of the MutSa, in Figure 3. The homeologous gap-repair (HeGR) MutSb, MutLa, and Rad1-Rad10 complexes in gap efficiency was reduced 2.4-fold relative to that between repair, we also examined the effects of the Srs2 and homologous sequences. Whereas the HGR events were Sgs1 helicases. While mutants defective in either heli- evenly distributed between NCO and CO events, only case exhibit a spontaneous hyperrecombination phe- 17% of the HeGR events were of the CO type. This notype (see Fabre et al. 2002), srs2 mutants exhibit a translates into a 7.3-fold reduction in homeologous hypo-rec phenotype when recombination is initiated relative to homologous CO events, but only a 1.4-fold with a DSB (Aylon et al. 2003; Ira et al. 2003). With reduction in NCO events. Thus, at least in the gap- regard to both spontaneous and DSB-initiated recom- repair assay used here, low levels of sequence divergence bination, loss of either Srs2 or Sgs1 shifts the distribu- have a strong inhibitory effect only on the maturation of tion of recombinants toward more CO events (Ira et al. recombination intermediates into CO products. The 1256 C. Welz-Voegele and S. Jinks-Robertson observation that the reduction of homeologous COs was not accompanied by a compensatory gain in NCOs suggests that not all repair events can be simply diverted from a CO to a NCO pathway. Regulation of homeologous gap repair: The roles of individual proteins in regulating the fidelity of recombination, and thereby reducing interactions between diverged sequences, were examined by transforming the gapped plasmid into an msh2, msh3, msh6, mlh1, pms1, rad1, srs2,orsgs1 strain. To correct for nonspecific effects of a given protein on the recombination process (e.g., its loss conferring a general hyper-rec phenotype or altering the CO–NCO distribution) the ratio of total HeGR to HGR was calculated in each strain background, as well as the ratio for NCO and CO events (Figure 4). The smaller the HeGR/HGR ratio, the greater the inhibitory effect of sequence divergence on the recombination process being examined; a ratio of 1.0 indicates equivalent efficiencies of homologous and homeologous recombination. For the WT strain, the baseline HeGR/HGR ratios were 0.41, 0.73, and 0.13 for total, NCO, and CO events, respectively. Relative to these baseline WT values, an increase in the HeGR/HGR ratio in a given mutant background indicates a relaxation of homology requirements and, therefore, a role of the corresponding protein in enforcing recombination fidelity. All of the potential mismatches generated during repair of the gapped plasmid are base–base mismatches and hence should primarily be detected by the MutSa complex. In accord with this prediction, the total or CO HeGR/HGR ratios changed little, if any, in an msh3 mutant, but increased to 1.0 in an msh6 or mlh1 mutant. The absence of an effect of sequence divergence on gap repair in the msh6 or mlh1 mutant is consistent with antirecombination activity being derived solely from the MMR machinery. In contrast to the msh6 and mlh1 mutants, the total HeGR/HGR ratio increased to 0.69 and the CO ratio to only 0.38 in an msh2 mutant; the transformation data in Table 1 indicate that these ratios indeed reflect a persistent reduction of homeologous recombination in the absence of Msh2. We suggest that this might reflect the role of MutSb in Figure 4.—Effect of sequence divergence on gap repair in stabilizing the substrate of the Rad1-Rad10 endonucle- different genetic backgrounds. The HeGR efficiency (His1/ ase, a role that could become more important when the Leu1) ratio was normalized to the HGR efficiency obtained interacting sequences are not identical. Finally, it in the same genetic background. The NCO and CO efficiencies were determined by multiplying the total repair efficiency by should be noted that the HeGR/HGR ratio in the the proportion of the relevant event and these efficiencies were pms1 mutant was only 0.77; the data in Table 1 again used to calculate the corresponding HeGR/HGR ratio. If se- suggest a persistent inhibition of homeologous gap quence divergence has no effect on repair efficiency, then repair when Pms1 is absent but not when Mlh1 is absent. HeGR/HGR ¼ 1. An HeGR/HRG ratio ,1 indicates that se- One possibility is that in the absence of Pms1, Mlh1 quence divergence inhibits repair; the smaller the ratio, the ang greater the inhibition. The double arrowheads to the right in- might partner with either Mlh2 or Mlh3 (W et al. dicate the magnitude of the inhibition in the WT background. 1999) to carry out a low level of antirecombination. In the HGR assay, loss of Rad1 was associated with a proteins in the regulation of recombination fidelity strong reduction in the proportion of CO events, while should be revealed as an increase in the HeGR/HGR loss of Sgs1 or Srs2 elevated the proportion of COs ratio for CO events in the appropriate mutant strains. (Table 1 and Figure 2). Additional roles for these Loss of either Sgs1 or Srs2 was associated with an Sequence Divergence and Recombination 1257 increase in the HeGR/HGR ratio; there was a 3.3-fold cleavage of both junctions in the same direction (both increase in the CO ratio in the sgs1 mutant, and a horizontally or both vertically) produces NCOs (step D), smaller, 1.9-fold increase in the srs2 mutant. A mis- while cleavage in different directions (one junction match-related antirecombination role for Sgs1 has been horizontally and the other vertically) produces COs previously observed in both spontaneous and DSB- (step E). Instead of HJ resolution by direct cleavage, induced recombination assays (Myung et al. 2001; the two junctions can migrate toward each other, with Spell and Jinks-Robertson 2004; Sugawara et al. the resulting hemicatenated molecules being resolved 2004), but this is the first indication that there may be by topoisomerase activity (step F). This latter mode of a similar activity associated with Srs2. In a rad1 back- resolution yields only NCO products and is thought to ground, there was no obvious change in the HeGR/ require the Sgs1 helicase and Top3 in yeast (Ira et al. HGR ratio, although an effect would have been difficult 2003). While capture of the unengaged end by an intact to detect given the very strong dependence of COs on D-loop leads to formation of a double HJ, cleavage of the the presence of Rad1. We note that this is in contrast to D-loop at its base results in the formation of a single HJ the clear role for the Rad1-Rad10 complex in limiting (step C). A partial dependence of COs on the Rad1 recombination between diverged, chromosomal in- endonuclease in some assays suggests that such cleavage verted-repeat substrates (Nicholson et al. 2000). can facilitate D-loop capture and/or HJ formation (Symington et al. 2000). As with a double HJ, the direction of cleaving a single HJ determines the NCO DISCUSSION or CO outcome; cleavage of the initially exchanged or The genetic regulation of mitotic recombination fidel- nonexchanged strands yields a NCO or CO product, ity was examined by transforming a gapped plasmid into respectively. The Sgs1 helicase might also be expected to strains containing either a 100%-identical homologous generate NCOs by reverse branch migration of a single or a 98%-identical homeologous chromosomal repair HJ intermediate. In contrast to the dissolution of double template. A key feature of the gap-repair system used HJs, however, no accompanying topoisomerase activity here is that it allows a distinction to be made between would be needed to resolve a single HJ. Finally, as an NCO and CO events; this was not possible with the alternative to second-end capture and HJ formation, inverted-repeat (IR) assay we previously used (Chen and collapse of the D-loop will free the extended 39 end to Jinks-Robertson 1998). An additional advantage of a pair with the unengaged, single-stranded tail on the gap-repair system is that both the position and nature of other side of the DSB/gap (step G). This latter type of the initiating lesion are known, whereas the IR assay recombination is referred to as synthesis-dependent only detects randomly initiated events. The major results strand annealing (SDSA) and yields only NCO products obtained using the gap-repair assay and discussed further (Paques and Haber 1999). The observed temporal below are that (1) the Rad1-Rad10 endonuclease strongly separation of NCO and CO events (Allers and Lichten promotes CO events, (2) sequence divergence affects CO 2001; Ira et al. 2003) is consistent with an SDSA pathway much more than NCO events, (3) the negative effect of that does not involve HJ resolution. In yeast, SDSA sequence divergence on recombination requires MutS- appears to be enhanced by the Srs2 helicase (Ira et al. and MutL-like complexes to similar extents, and (4) the 2003), which recently has been shown to dismantle Sgs1 and Srs2 helicases have roles in enforcing recombi- Rad51-containing D-loops in vitro (Dupaigne et al. nation identity requirements as well as in regulating the 2008). Although not applicable to the current gap-repair CO–NCO outcome. assay, single-strand annealing (SSA) is a final DSB repair In discussing the implications of results reported here, mechanism that specifically deletes the region between it is important to consider them in the context of current direct repeats and has been used to examine the regu- models of DSB repair (for a review, see Krogh and lation of recombination fidelity (Sugawara et al. 2004). Symington 2004). As shown in Figure 5, the ends of a Genetic control of gap repair between identical broken or gapped molecule are first resected to produce sequences: In a WT background with identical sub- 39 single-stranded tails that are incorporated into Rad51 strates, approximately one-half of the repaired plasmids nucleoprotein filaments. Following the invasion of a were integrated into the yeast genome. Neither the gap- homologous duplex by a nucleoprotein filament and repair efficiency nor the distribution of NCO–CO displacement of a D-loop, the invading 39 end is used to products was altered in msh6, mlh1,orpms1 mutants, prime new DNA synthesis (step A). When DNA synthesis indicating that the MutSa and MutLa complexes are proceeds past the other side of the DSB/gap, the not involved in recombination between identical se- unengaged 39 end can be ‘‘captured’’ by annealing to quences. In contrast, there was a specific, 4-fold de- the displaced D-loop, generating an intermediate with a crease in CO events in an msh3 mutant, implicating double HJ (step B). Double HJs are a key element of the MutSb in the processing of recombination intermedi- classic DSB repair model of recombination (Szostak ates. Plasmid integration was decreased even more (16- et al. 1983), and their mode of resolution can generate fold) in a rad1 strain, consistent with MutSb playing an either a CO or NCO product (steps D–E). Enzymatic accessory role by stabilizing the relevant Rad1-Rad10 1258 C. Welz-Voegele and S. Jinks-Robertson substrate (Surtees and Alani 2006). The strong de- may reflect the requirement of Srs2 for efficient re- pendence of COs on Rad1 is consistent with the covery from checkpoint-mediated cell-cycle arrest fol- suggestion that Rad1-Rad10 mediated D-loop cleavage lowing successful DSB repair (Vaze et al. 2002). A final promotes second-end capture to stabilize an HJ in- phenotype of sgs1 or srs2 mutants is an increased mitotic termediate (Symington et al. 2000). If this is the case, CO/NCO ratio for both spontaneous (Robert et al. then most COs in this system likely derive from a single 2006) and HO-initiated events (Ira et al. 2003; Lo et al. rather than a double HJ intermediate. Although there 2006). A similar effect was evident in our gap-repair appears to be a consistent role of Rad1-Rad10 in pro- system, where the CO/NCO ratio was 1.1, 6.7, and 2.0 in moting COs in plasmid-based DSB/gap repair assays WT, sgs1, and srs2 strains, respectively (Table 1). It has (Schiestl and Prakash 1988; Bartsch et al. 2000), it been suggested that the elevated CO/NCO ratio in srs2 should be noted that its effect during the repair of an mutants reflects a less efficient dismantling of D-loops HO-induced chromosomal DSB has been variable (Ira (Dupaigne et al. 2008) and hence loss of the NCO- et al. 2003; Nicholson et al. 2006). This variability could specific SDSA pathway (Figure 5; Ira et al. 2003). The be related to the lengths of the homology that flank the reduction in total gap repair in an srs2 mutant further DSB, which would limit the extent and hence stability of suggests that the SDSA pathway is not completely heteroduplex intermediates. Finally, the decrease in the interchangeable with the HJ pathways; that is, not all total efficiency of gap repair in msh3 or rad1 mutants of the persistent D-loops necessarily lead to the forma- suggests either that the Rad1-Rad10 complex plays a tion of HJs. Finally, the occurrence of more COs than role in the alternative SDSA pathway as well or that some NCOs in the srs2 mutant, where the non-SDSA pathways D-loop intermediates are dead-end products. A possible dominate, implies that the cleavage of HJs does not role of Rad1-Rad10 in SDSA might be in the removal of occur randomly, but rather in a manner that favors COs. 39 ends that have replicated past the region of plasmid- As an explanation for the elevated CO/NCO ratio in chromosome homology, an activity that has also been sgs1 mutants, it has been suggested that Sgs1 promotes a attributed to the Mus81-Mms4 complex (De Los Santos topoisomerase-mediated mode of dHJ resolution that et al. 2001; Fabre et al. 2002). yields only NCOs (Wu and Hickson 2003); in its In spontaneous recombination assays, elimination of absence the only option would be the enzymatic cleav- either Sgs1 or Srs2 results in a hyper-rec phenotype. Sgs1 age of both HJs (see Ira et al. 2003). Because COs in the is thought to reduce the accumulation of recombina- current gap-repair assay are strongly Rad1 dependent, tion-initiating lesions (Fabre et al. 2002) while Srs2 is we assume that most are generated through a single HJ thought to antagonize the formation of Rad51 nucleo- intermediate. The loss of 80% of the NCO events in the protein filaments (Krejci et al. 2003; Veauteet al. 2003). sgs1 mutant implies that Sgs1 might also dismantle If the primary inhibitory role of Srs2 derives from its single HJs and that, at least in our system, the major ability to strip Rad51 from nucleoprotein filaments before route of generating NCOs may be via an HJ-containing the initial strand invasion occurs, then it is not clear why pathway rather than the SDSA pathway. Furthermore, this negative role should be limited only to spontane- the very high CO/NCO ratio in the sgs1 mutant again ously initiated events. An interesting possibility is that indicates that HJ cleavage in this system generates pre- Srs2 efficiently disrupts filaments formed within single- dominantly CO products. Although there may be subtle stranded gaps, which may initiate most spontaneous differences, the roles of Srs2 and Sgs1 in our gap-repair recombination (Lettier et al. 2006), but is relatively assay are generally consistent with those inferred pre- inefficient at removing Rad51 from the free 39 tails viously (Ira et al. 2003), providing additional support formed at the ends of DSBs. This would be consistent that transformation-associated gap repair accurately with Srs2 ‘‘channeling’’ damage-containing gaps into a mimics the repair of chromosomal DSBs in yeast. postreplication repair pathway (error-prone translesion Sequence divergence differentially affects CO and DNA synthesis or error-free template switching) rather NCO events: One of the most striking findings in the than into the Rad51-dependent homologous recombi- current study is that sequence divergence impedes CO nation pathway (reviewed by Wu and Hickson 2006). In events to a much greater extent than NCO events. In the the case of a DSB, neither template switching nor gap gap-repair system used here, 2% sequence divergence filling by the translesion synthesis pathway would be a reduced CO events approximately sevenfold, but NCOs viable repair option. less than twofold. This difference is easiest to reconcile if The provision of initiating DSBs has revealed addi- sequence divergence exerts its primary effect subse- tional, recombination-promoting roles of Sgs1 and Srs2, quent to the initial strand invasion step that is common with the corresponding mutant strains exhibiting re- to all pathways in Figure 5; otherwise, one would expect duced repair of chromosomal DSBs (Aylon et al. 2003; CO and NCO events to be similarly affected. It is pos- Ira et al. 2003). Consistent with these results, we ob- sible, for example, that mismatches are detected and served 50 or 25% reductions in total gap-repair effi- trigger antirecombination only after extension of the ciency in an srs2 or sgs1 background, respectively. In the invading 39 end has been initiated. As long as the end case of the srs2 mutant, the reduction in total gap repair extends far enough to pair with the single-stranded tail Sequence Divergence and Recombination 1259

Figure 5.—Models for recombinational repair of a gapped plasmid using chromosomal DNA as a template. Plasmid and chromosomal DNA are indicated as black and red lines, respectively. Arrowheads rep- resent 39 ends, and dotted lines correspond to newly- synthesized DNA. The colors of the dotted lines correspond to that of the template. Heteroduplex DNA forms adjacent to the original gap and is depicted as paired black and red lines. Details of the models are given in the text.

on the other side of the DSB/gap (a 60-bp gap in the lengths of the interacting sequences, as in the gap-repair assay used here), SDSA would not be affected. An alter- assay used here, or when there is some critical level of native explanation is that HJ formation (i.e., second-end divergence between the interacting sequences. Finally, capture) requires more extensive heteroduplex forma- the detection of a differential effect requires that one be tion than does SDSA, in which case CO intermediates able to directly compare homologous and homeologous would contain more of the mismatches that inhibit recombination. In virtually all other assays, the homol- recombination. Finally, potential mismatches may not ogous control substrates contain one or more potential differentially affect the SDSA and HJ pathways, but mismatches. Exceptions include our previous studies rather may alter the mode of HJ resolution, specifically using IR substrates, where CO and NCO cannot be biasing the outcome toward more NCO events. The distinguished (Chen and Jinks-Robertson 1998) and presence of mismatches could either favor the non- the HO-initiated system of Nicholson et al. (2006), crossover mode of HJ cleavage or could promote the which only detects COs. dissolution of HJs by Sgs1. In terms of biological sig- In our earlier studies using 350-bp chromosomal IR nificance, the differential control of NCO and CO substrates, recombination was exquisitely sensitive to events would have the net effect of allowing a non- potential mismatches, with a level of sequence diver- identical sequence to be used as a repair template for a gence comparable to that used here (2%) reducing broken chromosome, while at the same time limiting recombination 50-fold (Datta et al. 1997). We suggest potentially deleterious rearrangements due to interac- that this reflects either a basic difference in plasmid– tions between dispersed repeats. chromosome vs. chromosome–chromosome recombi- The differential effect of sequence divergence on CO nation (e.g., recombination may occur before the and NCO events has not been previously reported, and plasmid DNA becomes organized into chromatin), a there are several reasons why an effect may not have difference in the initiating event (single-strand gap vs. been evident. First, some assays are capable of detecting DSB) or a cell cycle-related timing issue (Nicholson only NCO or only CO events (e.g.,Nicholson et al. et al. 2006). 2006), which obviously precludes the detection of The MMR system regulates recombination fidelity differential effects. Second, there may be inherent during gap repair: In the current gap-repair assay, the differences in the mismatch sensitivity of spontaneously CO events were not inhibited by sequence homeology in initiated vs. DSB-induced recombination (see below). msh6 or mlh1 mutants. These data indicate that, as in Third, it is possible that a differential effect is evident other types of recombination assays (Selva et al. 1995; only when heteroduplex extension is limited by the Datta et al. 1997; Sugawara et al. 2004), the regulation 1260 C. Welz-Voegele and S. Jinks-Robertson of recombination fidelity derives primarily from activity elimination of Srs2 did not differentially affect homol- of the MMR system. In contrast to assays used previously, ogous and homeologous recombination (Spell and however, where antirecombination was only partially Jinks-Robertson 2004; Sugawara et al. 2004). While dependent on MutLa (Chen and Jinks-Robertson the mismatch-related antirecombination activity of Sgs1 1999; Nicholson et al. 2000; Spell and Jinks-Robertson could result from interaction with Msh6 and/or Mlh1 2003; Sugawara et al. 2004), the contributions of MutS (Pedrazzi et al. 2001, 2003), no interactions of Srs2 with and MutL homologs appear to be equivalent in the gap- MMR proteins have been reported. One possibility is repair system. The variable requirement for MutLa that the presence of MMR proteins retards the forma- during mismatch-triggered antirecombination could tion and/or extension of the initial Rad51-dependent reflect basic differences in the underlying recombina- strand-invasion intermediate (Worth et al. 1994), tion process and/or antirecombination mechanism. thereby making it a more efficient target for dismantling There are, in principle, several distinct steps at which by Srs2. the MMR system could exert antirecombination ac- Advantages and limitations of a gap-repair assay: tivity (reviewed by Surtees et al. 2004). Mismatch de- Results obtained using gap-repair assays indicate that tection could block or retardstrandexchange,itcould these assays faithfully recapitulate the repair of HO- interfere with branch migration that extends hetero- induced chromosomal DSBs. A basic question that has duplex DNA, or it could decrease the stability of re- yet to be fully resolved, however, is whether spontaneous combination intermediates (e.g., trigger reverse branch mitotic recombination typically initiates with a DSB or migration). with a single-strand nick/gap, although recent data The roles of the Sgs1 and Srs2 helicases in the fidelity support the latter (Lettier et al. 2006). The distinct of gap repair: In vitro data suggest that the primary fidelity differences observed with our IR system, where recombination-related role of the Srs2 helicase is to most events appear to involve sister chromatids (Chen dismantle Rad51 nucleoprotein filaments (Krejci et al. and Jinks-Robertson 1998), vs. the gap-repair assay 2003; Veaute et al. 2003) or D-loops (Dupaigne et al. would be consistent with spontaneous recombination 2008), while that of the Sgs1 is in the dissolution of being primarily a gap-filling process. Regardless of the Holliday junctions (see Ira et al. 2003). An in vivo rele- lesion that normally initiates mitotic recombination, vance of the Sgs1-mediated branch migration has been however, it is clear that meiotic recombination initiates recently questioned, however, as the helicase activity of with Spo11-generated DSBs (Paques and Haber 1999). Sgs1 does not appear to be required for the mitotic CO– It is possible that mitotic DSB/gap repair systems more NCO decision in yeast (Lo et al. 2006). As in previous accurately reflect meiotic than mitotic mechanisms of studies using either IR substrates (Myung et al. 2001; recombination. Spell and Jinks-Robertson 2004) or an HO-initiated The experiments reported here have demonstrated SSA assay (Sugawara et al. 2004), we observed a role for that sequence divergence inhibits mitotic COs to a Sgs1 in limiting gap repair between diverged substrates. greater extent than NCOs. Examination of the associ- As in the IR assay, the Sgs1 requirement during gap repair ated gene conversion tracts may reveal why the former was only partial. Although the CO-specific effect of are more sensitive to potential mismatches. In addition, sequence divergence in the gap-repair assay would be the sequencing of recombinants derived from the consistent with the HJs being the primary target for transformation of MMR-defective cells, in which het- Sgs1-mediated antirecombination, HJs are not an in- eroduplex intermediates persist, may provide an un- termediate in SSA, where the antirecombination activi- precedented view of DSB-repair intermediates and how ties of MMR proteins and Sgs1 appear to be equivalent specific proteins alter recombination mechanisms. Spe- (Sugawara et al. 2004). One possibility is that mismatch- cifically, each of the pathways depicted in Figure 5 pre- containing annealed strands are the relevant target for dicts different positions of heteroduplex DNA (hDNA). Sgs1; strand annealing not only generates the key in- With SDSA, hDNA should only be present on the termediate in the SSA pathway, but also is relevant to plasmid and only on one side of the original gap; with second-end interactions during both SDSA and HJ HJ cleavage, there should be hDNA on both sides of the formation. The requirement for the helicase activity of gap, with a single hDNA tract associated with each his3 Sgs1 in the regulation of recombination fidelity (Spell allele; and with Sgs1-mediated HJ dissolution, there and Jinks-Robertson 2004), but not necessarily in HJ again should be hDNA on both sides of the gap, but all resolution (Lo et al. 2006), also would be consistent with a hDNA should be associated with the plasmid-encoded non-HJ intermediate being the primary target of the HIS3 allele. Finally, the production of a gapped plasmid mismatch-related Sgs1 antirecombination activity. by PCR will allow the position and/or size of the ini- Unexpectedly, the HeGR/HGR ratio for CO events tiating gap to be systematically varied by simply chang- was elevated in an srs2 mutant, suggesting that there may ing the positions of the PCR primers. The position of be an MMR-related role for Srs2 in modulating the the gap within the region of homology and/or its size fidelity of recombination in the gap-repair system. This could affect the overall repair efficiency, the CO–NCO is in contrast to the IR and SSA systems, where decision, or MMR-directed antirecombination. Sequence Divergence and Recombination 1261

We are grateful to Tom Petes, Lucas Argueso, and members of the Higgins, D., S. Prakash,P.Reynolds,R.Polakowska,S.Weber Jinks-Robertson lab for helpful discussions and comments on et al., 1983 Isolation and characterization of the RAD3 gene the manuscript. This work was supported by grant GM-038464 from of Saccharomyces cerevisiae and inviability of rad3 deletion mutants. the National Institutes of Health. Proc. Natl. Acad. Sci. USA 80: 5680–5684. Inbar, O., B. Liefshitz,G.Bitan and M. Kupiec, 2000 The relation- ship between homology length and crossing over during the repair of a broken chromosome. J. Biol. Chem. 275: 30833–30838. Ira, G., A. Malkova,G.Liberi,M.Foiani and J. E. Haber, LITERATURE CITED 2003 Srs2 and Sgs1-Top3 suppress crossovers during double- strand break repair in yeast. Cell 115: 401–411. hn ornfeld agrelius ivingston A , B.-Y., K. J. D ,T.J.F and D. M. L , Jinks-Robertson, S., M. Michelitch and S. Ramcharan, 1993 Sub- 1988 Effect of limited homology on gene conversion in a Sac- strate length requirements for efficient mitotic recombination in charomyces cerevisiae plasmid recombination system. Mol. Cell. Saccharomyces cerevisiae. Mol. Cell. Biol. 13: 3937–3950. Biol. 8: 2442–2448. Johnson, R. E., G. K. Kovvali,S.N.Guzder,N.S.Amin,C.Holm llers ichten A , T., and M. L , 2001 Differential timing and control of et al., 1996 Evidence for involvement of yeast proliferating cell noncrossover and crossover recombination during meiosis. Cell nuclear antigen in DNA mismatch repair. J. Biol. Chem. 271: 106: 47–57. ylon iefshitz itan anin upiec 27987–27990. A , Y., B. L ,G.B -B and M. K , 2003 Mo- Kirkpatrick, D. T., and T. D. Petes, 1997 Repair of DNA loops in- lecular dissection of mitotic recombination in the yeast Saccharo- volves DNA-mismatch and nucleotide-excision repair proteins. myces cerevisiae. Mol. Cell. Biol. 23: 1403–1417. artsch ang ymington Nature 387: 929–931. B , S., L. E. K and L. S. S , 2000 RAD51 is re- Kramer, W., B. Fartmann and E. C. Ringbeck, 1996 Transcription quired for the repair of plasmid double-stranded DNA gaps from of mutS and mutL-homologous genes in Saccharomyces cerevisiae either plasmid or chromosomal templates. Mol. Cell. Biol. 20: during the cell cycle. Mol. Gen. Genet. 252: 275–283. 1194–1205. ramer ramer illiamson ogel hen inks obertson K , W., B. K ,M.S.W and S. F , 1989 Clon- C , W., and S. J -R , 1998 Mismatch repair proteins ing and nucleotide sequence of DNA mismatch repair gene PMS1 regulate heteroduplex formation during mitotic recombination from Saccharomyces cerevisiae: homology of PMS1 to procaryotic in yeast. Mol. Cell. Biol. 18: 6525–6537. MutL and HexB. J. Bacteriol. 171: 5339–5346. Chen, W., and S. Jinks-Robertson, 1999 The role of the mismatch Krejci, L., S. Van Komen,Y.Li,J.Villemain,M.S.Reddy et al., repair machinery in regulating mitotic and meiotic recombina- 2003 DNA helicase Srs2 disrupts the Rad51 presynaptic fila- tion between diverged sequences in yeast. Genetics 151: 1299– ment. Nature 423: 305–309. 1313. Krogh, B. O., and L. S. Symington, 2004 Recombination proteins Datta, A., A. Adjiri,L.New,G.F.Crouse and S. Jinks-Robertson, in yeast. Annu. Rev. Genet. 38: 233–271. 1996 Mitotic crossovers between diverged sequences are regu- Kunkel, T. A., and D. A. Erie, 2005 DNA mismatch repair. Annu. lated by mismatch repair proteins in Saccharomyces cerevisiae. Rev. Biochem. 74: 681–710. Mol. Cell. Biol. 16: 1085–1093. Langston, L. D., and L. S. Symington, 2005 Opposing roles for Datta, A., M. Hendrix,M.Lipsitch and S. Jinks-Robertson, 1997 Dual roles for DNA sequence identity and the mismatch DNA structure-specific proteins Rad1, Msh2, Msh3, and Sgs1 in yeast gene targeting. EMBO J. 24: 2214–2223. repair system in the regulation of mitotic crossing-over in yeast. ettier eng de ayolo rdeniz eid Proc. Natl. Acad. Sci. USA 94: 9757–9762. L , G., Q. F ,A.A. M ,N.E ,R.J.R et al., de los Santos, T., J. Loidl,B.Larkin and N. M. Hollingsworth, 2006 The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae. PLoS Genet. 2: 1773–1786. 2001 A role for MMS4 in the processing of recombination inter- o affett mit likeman terk mediates during meiosis in Saccharomyces cerevisiae. Genetics 159: L , Y. C., K. S. P ,O.A ,J.A.C ,R.S et al., 1511–1525. 2006 Sgs1 regulates gene conversion tract lengths and cross- Drummond, J. T., J. Genschel,E.Wolf and P. Modrich, overs independently of its helicase activity. Mol. Cell. Biol. 26: 4086–4094. 1997 DHFR/MSH3 amplification in methotrexate-resistant cells yung atta hen olodner alters the hMutSa/hMutSb ratio and reduces the efficiency of M , K., A. D ,C.C and R. D. K , 2001 SGS1, the base-basemismatchrepair.Proc.Natl.Acad.Sci.USA94: 10144– Saccharomyces cerevisiae homologue of BLM and WRN, suppresses 10149. genome instability and homeologous recombination. Nat. Dupaigne, P., C. Le Breton,F.Fabre,S.Gangloff,E.Le Cam et al., Genet. 27: 1–4. icholson abbri eeves rouse 2008 The Srs2 helicase activity is stimulated by Rad51 filaments N , A., R. M. F ,J.W.R and G. F. C , on dsDNA: implications for crossover incidence during mitotic 2006 The effects of mismatch repair and RAD1 genes on inter- recombination. Mol. Cell 29: 243–254. chromosomal crossover recombination in Saccharomyces cerevisiae. Earley, M. C., and G. F. Crouse, 1998 The role of mismatch repair Genetics 173: 647–659. icholson endrix inks obertson rouse in the prevention of base pair mutations in Saccharomyces cerevi- N , A., M. H ,S.J -R and G. F. C , siae. Proc. Natl. Acad. Sci. USA 95: 15487–15491. 2000 Regulation of mitotic homeologous recombination in Fabre, F., A. Chan,W.D.Heyer and S. Gangloff, 2002 Alternate yeast: functions of mismatch repair and nucleotide excision re- pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent pair genes. Genetics 154: 133–146. formation of toxic recombination intermediates from single- Orr-Weaver, T. L., and J. W. Szostak, 1983 Yeast recombination: stranded gaps created by DNA replication. Proc. Natl. Acad. the association between double-strand gap repair and crossing- Sci. USA 99: 16887–16892. over. Proc. Natl. Acad. Sci. USA 80: 4417–4421. Fishman-Lobell, J., and J. E. Haber, 1992 Removal of nonhomol- Paques, F., and J. E. Haber, 1999 Multiple pathways of recombina- ogous DNA ends in double-strand break recombination: the tion induced by double-strand breaks in Saccharomyces cerevisiae. role of the yeast ultraviolet repair gene RAD1. Science 258: 480– Microbiol. Mol. Biol. Rev. 63: 349–404. 484. Pedrazzi, G., C. Perrera,H.Blaser,P.Kuster,G.Marra et al., Gietz, R. D., and R. A. Woods, 2002 Transformation of yeast by lith- 2001 Direct association of Bloom’s syndrome gene product ium acetate/single-stranded carrier DNA/polyethylene glycol with the human mismatch repair protein MLH1. Nucleic Acids method. Methods Enzymol. 350: 87–96. Res. 29: 4378–4386. Haghnazari, E., and W. D. Heyer, 2004 The DNA damage check- Pedrazzi, G., C. Z. Bachrati,N.Selak,I.Studer,M.Petkovic et al., point pathways exert multiple controls on the efficiency and out- 2003 The Bloom’s syndrome helicase interacts directly with the come of the repair of a double-stranded DNA gap. Nucleic Acids human DNA mismatch repair protein hMSH6. Biol. Chem. 384: Res. 32: 4257–4268. 1155–1164. Harfe, B. D., and S. Jinks-Robertson, 2000 DNA mismatch repair Prakash, S., and L. Prakash, 2000 Nucleotide excision repair in and genetic instability. Annu. Rev. Genet. 34: 359–399. yeast. Mutat. Res. 451: 13–24. Hayden, M. S., and B. Byers, 1992 Minimal extent of homology re- Robert, T., D. Dervins,F.Fabre and S. Gangloff, 2006 Mrc1 and quired for completion of meiotic recombination in Saccharomyces Srs2 are major actors in the regulation of spontaneous crossover. cerevisiae. Dev. Genet. 13: 498–514. EMBO J. 25: 2837–2846. 1262 C. Welz-Voegele and S. Jinks-Robertson

Saparbaev, M., L. Prakash and S. Prakash, 1996 Requirement of plasmid gap repair in nuclease-deficient strains of Saccharomyces mismatch repair genes MSH2 and MSH3 in RAD1–RAD10 path- cerevisiae. Nucleic Acids Res. 28: 4649–4656. way of mitotic recombination in Saccharomyces cerevisiae. Genetics Szostak, J. W., T. L. Orr-Weaver,R.J.Rothstein and F. W. Stahl, 142: 727–736. 1983 The double-strand-break repair model for recombination. Schiestl, R. H., and S. Prakash, 1988 RAD1, an excision repair Cell 33: 25–35. gene of Saccharomyces cerevisiae, is also involved in recombination. Tishkoff, D. X., A. L. Boerger,P.Bertrand,N.Filosi,G.M.Gaida Mol. Cell. Biol. 8: 3619–3626. et al., 1997 Identification and characterization of Saccharomyces Selva, E. M., L. New,G.F.Crouse and R. S. Lahue, 1995 Mismatch cerevisiae EXO1, a gene encoding an exonuclease that interacts correction acts as a barrier to homeologous recombination in with MSH2. Proc. Natl. Acad. Sci. USA 94: 7487–7492. Saccharomyces cerevisiae. Genetics 139: 1175–1188. Umar, A., A. B. Buermeyer,J.A.Simon,D.C.Thomas,A.B.Clark Sherman, F., 1991 Getting started with yeast. Meth. Enzymol. 194: et al., 1996 Requirement for PCNA in DNA mismatch repair at a 3–20. step preceding DNA resynthesis. Cell 87: 65–73. Sikorski, R. S., and P. Hieter, 1989 A system of shuttle vectors and Vaze, M. B., A. Pellicioli,S.E.Lee,G.Ira,G.Liberi et al., yeast host strains designed for efficient manipulation of DNA in 2002 Recovery from checkpoint-mediated arrest after repair of Saccharomyces cerevisiae. Genetics 122: 19–27. a double-strand break requires Srs2 helicase. Mol. Cell 10: 373–385. Spell, R. M., and S. Jinks-Robertson, 2003 Role of mismatch re- Veaute, X., J. Jeusset,C.Soustelle,S.C.Kowalczykowski,E.Le pair in the fidelity of RAD51- and RAD59-dependent recombina- Cam et al., 2003 The Srs2 helicase prevents recombination by tion in Saccharomyces cerevisiae. Genetics 165: 1733–1744. disrupting Rad51 nucleoprotein filaments. Nature 423: 309–312. Spell, R. M., and S. Jinks-Robertson, 2004 Examination of the Wach, A., A. Brachat,R.Pohlmann and P. Philippsen, 1994 New roles of the Sgs1 and Srs2 helicases in the enforcement of recom- heterologous modules for classical or PCR-based gene disrup- bination fidelity in Saccharomyces cerevisiae. Genetics 168: 1855– tions in Saccharomyces cerevisiae. Yeast 10: 1793–1808. 1865. Wang, T.-F., N. Kleckner and N. Hunter, 1999 Functional spec- Stone, J., R. Gealy,T.Petes and S. Jinks-Robertson, 2008 Role of ificity of MutL homologs in yeast: evidence for three Mlh1-based PCNA interactions in the mismatch repair-dependent processing heterocomplexes with distinct roles during meiosis in recombi- of mitotic and meiotic recombination intermediates in yeast. Ge- nation and mismatch correction. Proc. Natl. Acad. Sci. USA 96: netics 178: 1221–1236. 13914–13919. Sugawara, N., T. Goldfarb,B.Studamire,E.Alani and J. E. Worth, L. J., S. Clark,M.Radman and P. Modrich, 1994 Mis- Haber, 2004 Heteroduplex rejection during single-strand an- match repair proteins MutS and MutL inhibit RecA-catalyzed nealing requires Sgs1 helicase and mismatch repair proteins strand transfer between diverged DNAs. Proc. Natl. Acad. Sci. Msh2 and Msh6 but not Pms1. Proc. Natl. Acad. Sci. USA 101: USA 91: 3238–3241. 9315–9320. Wu, L., and I. D. Hickson, 2003 The Bloom’s syndrome helicase Surtees, J. A., and E. Alani, 2006 Mismatch repair factor MSH2– suppresses crossing over during homologous recombination. Na- MSH3 binds and alters the conformation of branched DNA structure 426: 870–874. tures predicted to form during genetic recombination. J. Mol. Wu, L., and I. D. Hickson, 2006 DNA helicases required for homol- Biol. 360: 523–536. ogous recombination and repair of damaged replication forks. Surtees, J. A., J. L. Argueso and E. Alani, 2004 Mismatch repair Annu. Rev. Genet. 40: 279–306. proteins: key regulators of genetic recombination. Cytogenet. Genome Res. 107: 146–159. Symington, L. S., L. E. Kang and S. Moreau, 2000 Alteration of gene conversion tract length and associated crossing over during Communicating editor: E. Alani