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Eric Alani - One of the best experts on this subject based on the ideXlab platform.
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MLH1/3 variants causing aneuploidy, pregnancy loss, and premature reproductive aging
2021Co-Authors: Robert Fragoza, Eric Alani, Najla Al-sweel, Cecilia S. Blengini, Tina N Tran, Gianno Pannafino, Kerry J. Schimenti, Karen Schindler, John C. SchimentiAbstract:Most spontaneous pregnancy losses are a result of embryonic aneuploidy stemming from mis-segregation of chromosomes during meiosis. Proper disjunction of homologous chromosomes is dependent upon precise control of crossing-over, a process requiring the mismatch repair (MMR) genes MLH1 and MLH3. Both are required for fertility and completion of meiosis in mice. People inheriting variants in these genes are often at high risk for colorectal cancer and Lynch syndrome, yet the potential impacts of variants upon reproduction are unclear. To determine if MLH1/3 variants (namely single nucleotide polymorphisms, or SNPs) in human populations can cause reproductive abnormalities, we used a combination of computational predictions, yeast two-hybrid assays, and assays of MMR and recombination in yeast to select nine MLH1 and MLH3 variants for modeling in mice via genome editing. We identified 7 alleles that caused reproductive defects in mice including subfertility in females, male infertility, reduced sperm counts, and increased spermatocyte apoptosis. Remarkably, these alleles in females caused age-dependent decreases in litter size, and increased resorption of embryos during pregnancy. These outcomes were likely a consequence of reduced meiotic chiasmata, in turn causing an increase in misaligned chromosomes and univalents in meiotic metaphase I (MI). Our data indicate that segregating hypomorphic alleles of meiotic recombination genes in populations can predispose females to increased incidence of pregnancy loss from gamete aneuploidy.
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mutation of MLH3 endonuclease motif reveals integration between crossover pathways in mammalian meiosis
bioRxiv, 2019Co-Authors: Melissa Toledo, Vandana Raghavan, Xianfei Sun, Miguel A Brienoenriquez, Stephen K Gray, Jeffrey Pea, Anita Venkatesh, Lekha Patel, Peter L Borst, Eric AlaniAbstract:SUMMARY The MLH1-MLH3 complex is essential for crossing over in mammalian meiosis. We generated a mutation in mouse MLH3 that disrupts its conserved endonuclease domain and show that it disrupts crossing over in a manner distinct from the null, but also results in male infertility. ABSTRACT During meiotic prophase I, double strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). MLH3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse MLH3DN allele harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. MLH3DN/DN males, like fully null MLH3−/− males, have no spermatozoa and are infertile, yet spermatocytes have normal DSBs and undergo normal synapsis events in early prophase I. Unlike MLH3−/− males, however, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, DSB repair and CO designation factors persist in MLH3DN/DN males, indicating a temporal delay in repair events. While MLH3DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in MLH3−/− males (10%), suggesting that the allele may be partially functional or that other pathways can generate COs from these MutLγ-defined repair intermediates in MLH3DN/DN males, with evidence favoring the latter option. Double mutant mice that are homozygous for the MLH3DN/DN mutation along with a null allele of Mus81, show losses in chiasmata approaching levels observed in MLH3−/− males, suggesting that the MUS81-EME1-regulated crossover pathway accounts for some of the increased residual chiasmata observed in the MLH3DN/DN spermatocytes. These results demonstrate that an intact MLH3 endonuclease domain is essential for most COs in mammalian meiosis, and that an endonuclease-impaired MutLγ retains the ability to facilitate the recruitment of other repair pathways, including MUS81 -EME1.
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a mutation in the endonuclease domain of mouse MLH3 reveals novel roles for mutlγ during crossover formation in meiotic prophase i
bioRxiv, 2019Co-Authors: Melissa Toledo, Vandana Raghavan, Xianfei Sun, Miguel A Brienoenriquez, Stephen K Gray, Jeffrey Pea, Anita Venkatesh, Lekha Patel, Peter L Borst, Eric AlaniAbstract:During meiotic prophase I, double strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). MLH3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse MLH3DN allele harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. MLH3DN/DN males, like fully null MLH3-/- males, have no spermatozoa and are infertile, yet spermatocytes have normal DSBs and undergo normal synapsis events in early prophase I. Unlike MLH3-/- males, however, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, DSB repair and CO designation factors persist in MLH3DN/DN males, indicating a temporal delay in repair events. While MLH3DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in MLH3-/- males (10%), suggesting that the allele may be partially functional or that other pathways can generate COs from these MutLγ-defined repair intermediates in MLH3DN/DN males, with evidence favoring the latter option. Double mutant mice that are homozygous for the MLH3DN/DN mutation along with a null allele of Mus81, show losses in chiasmata approaching levels observed in MLH3-/- males, suggesting that the MUS81-EME1-regulated crossover pathway accounts for some of the increased residual chiasmata observed in the MLH3DN/DN spermatocytes. These results demonstrate that an intact MLH3 endonuclease domain is essential for most COs in mammalian meiosis, and that an endonuclease-impaired MutLγ retains the ability to facilitate the recruitment of other repair pathways, including MUS81-EME1.
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MLH3 mutations in baker s yeast alter meiotic recombination outcomes by increasing noncrossover events genome wide
PLOS Genetics, 2017Co-Authors: Najla Alsweel, Carol M Manhart, Jennifer A Surtees, Vandana Raghavan, Abhishek Dutta, V P Ajith, Luigi Di Vietro, Nabila Khondakar, K T Nishant, Eric AlaniAbstract:Mlh1-MLH3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-MLH3 functions in both meiosis and MMR, we analyzed in baker’s yeast 60 new MLH3 alleles. Five alleles specifically disrupted MMR, whereas one (MLH3-32) specifically disrupted meiotic crossing over. Mlh1-MLH3 representatives for each class were purified and characterized. Both Mlh1-MLH3-32 (MMR+, crossover-) and Mlh1-MLH3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-MLH3 in MMR, stimulated the endonuclease activity of Mlh1-MLH3-32 but not Mlh1-MLH3-45, suggesting that Mlh1-MLH3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for wild-type and MMR+ crossover-, MMR- crossover+, endonuclease defective and null MLH3 mutants in an S288c/YJM789 hybrid background. Compared to wild-type, all of the MLH3 mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. Our observations provide a structure-function map for MLH3 that reveals the importance of protein-protein interactions in regulating Mlh1-MLH3’s enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.
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the mismatch repair and meiotic recombination endonuclease mlh1 MLH3 is activated by polymer formation and can cleave dna substrates in trans
PLOS Biology, 2017Co-Authors: Carol M Manhart, Martin White, Joaquin Ortega, Jennifer A Surtees, Eric AlaniAbstract:Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-MLH3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-MLH3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-MLH3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-MLH3. In addition, Mlh1-MLH3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-MLH3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-MLH3 is loaded onto DNA to form an activated polymer that cleaves DNA.
Paula E Cohen - One of the best experts on this subject based on the ideXlab platform.
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Somatic CAG expansion in Huntington's disease is dependent on the MLH3 endonuclease domain, which can be excluded via splice redirection.
Nucleic acids research, 2021Co-Authors: Jennie C L Roy, Paula E Cohen, Ed Grabczyk, Antonia Vitalo, Marissa A Andrew, Eduarda Mota-silva, Marina Kovalenko, Zoe Burch, Anh M Nhu, Vanessa C WheelerAbstract:Somatic expansion of the CAG repeat tract that causes Huntington's disease (HD) is thought to contribute to the rate of disease pathogenesis. Therefore, factors influencing repeat expansion are potential therapeutic targets. Genes in the DNA mismatch repair pathway are critical drivers of somatic expansion in HD mouse models. Here, we have tested, using genetic and pharmacological approaches, the role of the endonuclease domain of the mismatch repair protein MLH3 in somatic CAG expansion in HD mice and patient cells. A point mutation in the MLH3 endonuclease domain completely eliminated CAG expansion in the brain and peripheral tissues of a HD knock-in mouse model (HttQ111). To test whether the MLH3 endonuclease could be manipulated pharmacologically, we delivered splice switching oligonucleotides in mice to redirect MLH3 splicing to exclude the endonuclease domain. Splice redirection to an isoform lacking the endonuclease domain was associated with reduced CAG expansion. Finally, CAG expansion in HD patient-derived primary fibroblasts was also significantly reduced by redirecting MLH3 splicing to the endogenous endonuclease domain-lacking isoform. These data indicate the potential of targeting the MLH3 endonuclease domain to slow somatic CAG repeat expansion in HD, a therapeutic strategy that may be applicable across multiple repeat expansion disorders.
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Somatic CAG expansion in Huntington's disease is dependent on the MLH3 endonuclease domain, which can be excluded via MLH3 splice redirection to suppress expansion
2020Co-Authors: Jennie C L Roy, Paula E Cohen, Antonia Vitalo, Marissa A Andrew, Eduarda Mota-silva, Marina Kovalenko, Zoe Burch, Anh M Nhu, E. Grabczyk, Vanessa C WheelerAbstract:Abstract Somatic expansion of the CAG repeat tract that causes Huntington’s disease (HD) is thought to contribute to the rate of disease pathogenesis. Therefore, factors influencing repeat expansion are potential therapeutic targets. Genes in the DNA mismatch repair pathway are critical drivers of somatic expansion in HD mouse models. Here, we have tested, using genetic and pharmacological approaches, the role of the endonuclease domain of the mismatch repair protein MLH3 in somatic CAG expansion in HD mice and patient cells. A point mutation in the MLH3 endonuclease domain completely eliminated CAG expansion in the brain and peripheral tissues of a HD knock-in mouse model (HttQ111). To test whether the MLH3 endonuclease could be manipulated pharmacologically, we delivered splice switching oligonucleotides in mice to redirect MLH3 splicing to exclude the endonuclease domain. Splice redirection to an isoform lacking the endonuclease domain was associated with reduced CAG expansion. Finally, CAG expansion in HD patient-derived primary fibroblasts was also significantly reduced by redirecting MLH3 splicing to the endogenous endonuclease domain-lacking isoform. These data indicate the potential of targeting the MLH3 endonuclease domain to slow somatic CAG repeat expansion in HD, a therapeutic strategy that may be applicable across multiple repeat expansion disorders.
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mus81 generates a subset of mlh1 MLH3 independent crossovers in mammalian meiosis
PLOS Genetics, 2008Co-Authors: Kim J Holloway, James G Booth, Clare H Mcgowan, Paula E CohenAbstract:Two eukaryotic pathways for processing double-strand breaks (DSBs) as crossovers have been described, one dependent on the MutL homologs Mlh1 and MLH3, and the other on the structure-specific endonuclease Mus81. Mammalian MUS81 has been implicated in maintenance of genomic stability in somatic cells; however, little is known about its role during meiosis. Mus81-deficient mice were originally reported as being viable and fertile, with normal meiotic progression; however, a more detailed examination of meiotic progression in Mus81-null animals and WT controls reveals significant meiotic defects in the mutants. These include smaller testis size, a depletion of mature epididymal sperm, significantly upregulated accumulation of MLH1 on chromosomes from pachytene meiocytes in an interference-independent fashion, and a subset of meiotic DSBs that fail to be repaired. Interestingly, chiasmata numbers in spermatocytes from Mus81−/− animals are normal, suggesting additional integrated mechanisms controlling the two distinct crossover pathways. This study is the first in-depth analysis of meiotic progression in Mus81-nullizygous mice, and our results implicate the MUS81 pathway as a regulator of crossover frequency and placement in mammals.
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MUS81 Generates a Subset of MLH1-MLH3–Independent Crossovers in Mammalian Meiosis
PLoS genetics, 2008Co-Authors: J. Kim Holloway, Winfried Edelmann, James G Booth, Clare H Mcgowan, Paula E CohenAbstract:Two eukaryotic pathways for processing double-strand breaks (DSBs) as crossovers have been described, one dependent on the MutL homologs Mlh1 and MLH3, and the other on the structure-specific endonuclease Mus81. Mammalian MUS81 has been implicated in maintenance of genomic stability in somatic cells; however, little is known about its role during meiosis. Mus81-deficient mice were originally reported as being viable and fertile, with normal meiotic progression; however, a more detailed examination of meiotic progression in Mus81-null animals and WT controls reveals significant meiotic defects in the mutants. These include smaller testis size, a depletion of mature epididymal sperm, significantly upregulated accumulation of MLH1 on chromosomes from pachytene meiocytes in an interference-independent fashion, and a subset of meiotic DSBs that fail to be repaired. Interestingly, chiasmata numbers in spermatocytes from Mus81−/− animals are normal, suggesting additional integrated mechanisms controlling the two distinct crossover pathways. This study is the first in-depth analysis of meiotic progression in Mus81-nullizygous mice, and our results implicate the MUS81 pathway as a regulator of crossover frequency and placement in mammals.
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Distinct functions of MLH3 at recombination hot spots in the mouse.
Genetics, 2008Co-Authors: Anton Svetlanov, Paula E Cohen, Frédéric Baudat, Bernard De MassyAbstract:The four mammalian MutL homologs (MLH1, MLH3, PMS1, and PMS2) participate in a variety of events, including postreplicative DNA repair, prevention of homeologous recombination, and crossover formation during meiosis. In this latter role, MLH1–MLH3 heterodimers predominate and are essential for prophase I progression. Previous studies demonstrated that mice lacking Mlh1 exhibit a 90% reduction in crossing over at the Psmb9 hot spot while noncrossovers, which do not result in exchange of flanking markers but arise from the same double-strand break event, are unaffected. Using a PCR-based strategy that allows for detailed analysis of crossovers and noncrossovers, we show here that MLH3−/− exhibit a 85–94% reduction in the number of crossovers at the Psmb9 hot spot. Most of the remaining crossovers in MLH3−/− meiocytes represent simple exchanges similar to those seen in wild-type mice, with a small fraction (6%) representing complex events that can extend far from the initiation zone. Interestingly, we detect an increase of noncrossovers in MLH3−/− spermatocytes. These results suggest that MLH3 functions predominantly with MLH1 to promote crossovers, while noncrossover events do not require these activities. Furthermore, these results indicate that ∼10% of crossovers in the mouse are independent of MLH3, suggesting the existence of alternative crossover pathways in mammals.
Neal Sugawara - One of the best experts on this subject based on the ideXlab platform.
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heteroduplex rejection during single strand annealing requires sgs1 helicase and mismatch repair proteins msh2 and msh6 but not pms1
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Neal Sugawara, Barbara Studamire, Tamara Goldfarb, Eric AlaniAbstract:Recombination between moderately divergent DNA sequences is impaired compared with identical sequences. In yeast, an HO endonuclease-induced double-strand break can be repaired by single-strand annealing (SSA) between flanking homologous sequences. A 3% sequence divergence between 205-bp sequences flanking the double-strand break caused a 6-fold reduction in repair compared with identical sequences. This reduction in heteroduplex rejection was suppressed in a mismatch repair-defective msh6Δ strain and partially suppressed in an msh2 separation-of-function mutant. In mlh1Δ strains, heteroduplex rejection was greater than in msh6Δ strains but less than in wild type. Deleting PMS1, MLH2,or MLH3 had no effect on heteroduplex rejection, but a pms1Δ mlh2Δ MLH3Δ triple mutant resembled mlh1Δ. However, correction of the mismatches within heteroduplex SSA intermediates required PMS1 and MLH1 to the same extent as MSH2 and MSH6. An SSA competition assay in which either diverged or identical repeats can be used for repair showed that heteroduplex DNA is likely to be unwound rather than degraded. This conclusion is supported by the finding that deleting the SGS1 helicase also suppressed heteroduplex rejection.
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Heteroduplex rejection during single-strand annealing requires Sgs1 helicase and mismatch repair proteins Msh2 and Msh6 but not Pms1
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Neal Sugawara, Barbara Studamire, Tamara Goldfarb, Eric Alani, James E. HaberAbstract:Recombination between moderately divergent DNA sequences is impaired compared with identical sequences. In yeast, an HO endonuclease-induced double-strand break can be repaired by single-strand annealing (SSA) between flanking homologous sequences. A 3% sequence divergence between 205-bp sequences flanking the double-strand break caused a 6-fold reduction in repair compared with identical sequences. This reduction in heteroduplex rejection was suppressed in a mismatch repair-defective msh6 Delta strain and partially suppressed in an msh2 separation-of-function mutant. In mlh1 Delta strains, heteroduplex rejection was greater than in msh6 Delta strains but less than in wild type. Deleting PMS1, MLH2,or MLH3 had no effect on heteroduplex rejection, but a pms1 Delta mlh2 Delta MLH3 Delta triple mutant resembled mlh1 Delta. However, correction of the mismatches within heteroduplex SSA intermediates required PMS1 and MLH1 to the same extent as MSH2 and MSH6. An SSA competition assay in which either diverged or identical repeats can be used for repair showed that heteroduplex DNA is likely to be unwound rather than degraded. This conclusion is supported by the finding that deleting the SGS1 helicase also suppressed heteroduplex rejection.
James E. Haber - One of the best experts on this subject based on the ideXlab platform.
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Heteroduplex rejection during single-strand annealing requires Sgs1 helicase and mismatch repair proteins Msh2 and Msh6 but not Pms1
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Neal Sugawara, Barbara Studamire, Tamara Goldfarb, Eric Alani, James E. HaberAbstract:Recombination between moderately divergent DNA sequences is impaired compared with identical sequences. In yeast, an HO endonuclease-induced double-strand break can be repaired by single-strand annealing (SSA) between flanking homologous sequences. A 3% sequence divergence between 205-bp sequences flanking the double-strand break caused a 6-fold reduction in repair compared with identical sequences. This reduction in heteroduplex rejection was suppressed in a mismatch repair-defective msh6 Delta strain and partially suppressed in an msh2 separation-of-function mutant. In mlh1 Delta strains, heteroduplex rejection was greater than in msh6 Delta strains but less than in wild type. Deleting PMS1, MLH2,or MLH3 had no effect on heteroduplex rejection, but a pms1 Delta mlh2 Delta MLH3 Delta triple mutant resembled mlh1 Delta. However, correction of the mismatches within heteroduplex SSA intermediates required PMS1 and MLH1 to the same extent as MSH2 and MSH6. An SSA competition assay in which either diverged or identical repeats can be used for repair showed that heteroduplex DNA is likely to be unwound rather than degraded. This conclusion is supported by the finding that deleting the SGS1 helicase also suppressed heteroduplex rejection.
Barbara Studamire - One of the best experts on this subject based on the ideXlab platform.
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heteroduplex rejection during single strand annealing requires sgs1 helicase and mismatch repair proteins msh2 and msh6 but not pms1
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Neal Sugawara, Barbara Studamire, Tamara Goldfarb, Eric AlaniAbstract:Recombination between moderately divergent DNA sequences is impaired compared with identical sequences. In yeast, an HO endonuclease-induced double-strand break can be repaired by single-strand annealing (SSA) between flanking homologous sequences. A 3% sequence divergence between 205-bp sequences flanking the double-strand break caused a 6-fold reduction in repair compared with identical sequences. This reduction in heteroduplex rejection was suppressed in a mismatch repair-defective msh6Δ strain and partially suppressed in an msh2 separation-of-function mutant. In mlh1Δ strains, heteroduplex rejection was greater than in msh6Δ strains but less than in wild type. Deleting PMS1, MLH2,or MLH3 had no effect on heteroduplex rejection, but a pms1Δ mlh2Δ MLH3Δ triple mutant resembled mlh1Δ. However, correction of the mismatches within heteroduplex SSA intermediates required PMS1 and MLH1 to the same extent as MSH2 and MSH6. An SSA competition assay in which either diverged or identical repeats can be used for repair showed that heteroduplex DNA is likely to be unwound rather than degraded. This conclusion is supported by the finding that deleting the SGS1 helicase also suppressed heteroduplex rejection.
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Heteroduplex rejection during single-strand annealing requires Sgs1 helicase and mismatch repair proteins Msh2 and Msh6 but not Pms1
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Neal Sugawara, Barbara Studamire, Tamara Goldfarb, Eric Alani, James E. HaberAbstract:Recombination between moderately divergent DNA sequences is impaired compared with identical sequences. In yeast, an HO endonuclease-induced double-strand break can be repaired by single-strand annealing (SSA) between flanking homologous sequences. A 3% sequence divergence between 205-bp sequences flanking the double-strand break caused a 6-fold reduction in repair compared with identical sequences. This reduction in heteroduplex rejection was suppressed in a mismatch repair-defective msh6 Delta strain and partially suppressed in an msh2 separation-of-function mutant. In mlh1 Delta strains, heteroduplex rejection was greater than in msh6 Delta strains but less than in wild type. Deleting PMS1, MLH2,or MLH3 had no effect on heteroduplex rejection, but a pms1 Delta mlh2 Delta MLH3 Delta triple mutant resembled mlh1 Delta. However, correction of the mismatches within heteroduplex SSA intermediates required PMS1 and MLH1 to the same extent as MSH2 and MSH6. An SSA competition assay in which either diverged or identical repeats can be used for repair showed that heteroduplex DNA is likely to be unwound rather than degraded. This conclusion is supported by the finding that deleting the SGS1 helicase also suppressed heteroduplex rejection.