In Saccharomyces cerevisiae the MSH4-MSH5, MLH1-MLH3, and MUS81-MMS4 complexes act to promote crossing over during meiosis. MSH4-MSH5, but not MUS81-MMS4, promotes crossovers that display interference. A role for MLH1-MLH3 in crossover control is less clear partly because mlh1Δ mutants retain crossover interference yet display a decrease in crossing over that is only slightly less severe than that seen in msh4Δ and msh5Δ mutants. We analyzed the effects of msh5Δ, mlh1Δ, and mms4Δ single, double, and triple mutants on meiotic crossing over at four consecutive genetic intervals on chromosome XV using newly developed computer software. mlh1Δ mms4Δ double mutants displayed the largest decrease in crossing over (13- to 15-fold) of all mutant combinations, yet these strains displayed relatively high spore viability (42%). In contrast, msh5Δ mms4Δ and msh5Δ mms4Δ mlh1Δ mutants displayed smaller decreases in crossing over (4- to 6-fold); however, spore viability (18–19%) was lower in these strains than in mlh1Δ mms4Δ strains. These data suggest that meiotic crossing over can occur in yeast through three distinct crossover pathways. In one pathway, MUS81-MMS4 promotes interference-independent crossing over; in a second pathway, both MSH4-MSH5 and MLH1-MLH3 promote interference-dependent crossovers. A third pathway, which appears to be repressed by MSH4-MSH5, yields deleterious crossovers.

Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair.

The yeast Saccharomyces cerevisiae encodes six proteins, Msh1p to Msh6p, that show strong amino acid sequence similarity to MutS, a central component of the bacterial mutHLS mismatch repair system. Recent studies with humans and S. cerevisiae suggest that in eukaryotes, specific MutS homolog complexes that display unique DNA mismatch specificities exist. In this study, the S. cerevisiae 109-kDa Msh2 and 140-kDa Msh6 proteins were cooverexpressed in S. cerevisiae and shown to interact in an immunoprecipitation assay and by conventional chromatography. Deletion analysis of MSH2 indicated that the carboxy-terminal 114 amino acids of Msh2p are important for Msh6p interaction. Purified Msh2p-Msh6p selectively bound to duplex oligonucleotide substrates containing a G/T mismatch and a +1 insertion mismatch but did not show specific binding to +2 and +4 insertion mismatches. The mismatch binding specificity of the Msh2p-Msh6p complex, as measured by on-rate and off-rate binding studies, was abolished by ATP. Interestingly, palindromic substrates that are poorly repaired in vivo were specifically recognized by Msh2p-Msh6p; however, the binding of Msh2p-Msh6p to these substrates was not modulated by ATP. Taken together, these studies suggest that the repair of a base pair mismatch by the Msh2p-Msh6p complex is dependent on the ability of the Msh2p-Msh6p-DNA mismatch complex to use ATP hydrolysis to activate downstream events in mismatch repair.

Interference-dependent crossing over in yeast and mammalian meioses involves the mismatch repair protein homologs MSH4-MSH5 and MLH1-MLH3. The MLH3 protein contains a highly conserved metal-binding motif DQHA(X)₂E(X)₄E that is found in a subset of MLH proteins predicted to have endonuclease activities (Kadyrovet al. 2006). Mutations within this motif in human PMS2 and Saccharomyces cerevisiae PMS1 disrupted the endonuclease and mismatch repair activities of MLH1-PMS2 and MLH1-PMS1, respectively (Kadyrovet al. 2006, 2007; Erdenizet al. 2007). As a first step in determining whether such an activity is required during meiosis, we made mutations in the MLH3 putative endonuclease domain motif (-D523N, -E529K) and found that single and double mutations conferred mlh3-null-like defects with respect to meiotic spore viability and crossing over. Yeast two-hybrid and chromatography analyses showed that the interaction between MLH1 and mlh3-D523N was maintained, suggesting that the mlh3-D523N mutation did not disrupt the stability of MLH3. The mlh3-D523N mutant also displayed a mutator phenotype in vegetative growth that was similar to mlh3Δ. Overexpression of this allele conferred a dominant-negative phenotype with respect to mismatch repair. These studies suggest that the putative endonuclease domain of MLH3 plays an important role in facilitating mismatch repair and meiotic crossing over.

Chromosome movements are a general feature of mid-prophase of meiosis. In budding yeast, meiotic chromosomes exhibit dynamic movements, led by nuclear envelope (NE)-associated telomeres, throughout the zygotene and pachytene stages. Zygotene motion underlies the global tendency for colocalization of NE-associated chromosome ends in a “bouquet.” In this study, we identify Csm4 as a new molecular participant in these processes and show that, unlike the two previously identified components, Ndj1 and Mps3, Csm4 is not required for meiosis-specific telomere/NE association. Instead, it acts to couple telomere/NE ensembles to a force generation mechanism. Mutants lacking Csm4 and/or Ndj1 display the following closely related phenotypes: (i) elevated crossover (CO) frequencies and decreased CO interference without abrogation of normal pathways; (ii) delayed progression of recombination, and recombination-coupled chromosome morphogenesis, with resulting delays in the MI division; and (iii) nondisjunction of homologs at the MI division for some reason other than absence of (the obligatory) CO(s). The recombination effects are discussed in the context of a model where the underlying defect is chromosome movement, the absence of which results in persistence of inappropriate chromosome relationships that, in turn, results in the observed mutant phenotypes. In meiosis, cells specified to become gametes (eggs or sperm) undergo a single round of DNA replication followed by two consecutive chromosomal divisions. In most organisms, the proper segregation of chromosomes at the first meiotic division is mechanically dependent upon genetic exchange, or crossing over, at homologous sites along chromosomes. This process is highly regulated so that every pair of matched chromosomes, regardless of size, receives at least one crossover. In humans, defects in this recombination process can lead to a variety of chromosome aneuploidy syndromes. During early stages in meiosis, the ends of chromosomes, called telomeres, associate with the envelope of the nucleus and undergo highly dynamic movements. We identified a new component of the movement-generating system, Csm4, in budding yeast. In the absence of Csm4, the telomeres associate with the nuclear envelope but are locked in an immobile state. In addition, strains lacking Csm4 show delayed recombination progression and high levels of chromosome mis-segregation at the first meiotic division. These findings suggest that, during meiosis, Csm4 is involved in coupling telomere complexes to the movement-generating system and that chromosome motion is important for the completion of early steps in recombination.

Mismatch repair (MMR) systems correct DNA mismatches that result from DNA polymerase misincorporation errors. Mismatches also appear in heteroduplex DNA intermediates formed during recombination between nearly identical sequences, and can be corrected by MMR or removed through an unwinding mechanism, known as anti-recombination or heteroduplex rejection. We review studies, primarily in baker's yeast, which support how specific factors can regulate the MMR/anti-recombination decision. Based on recent advances, we present models for how DNA structure, relative amounts of key repair proteins, the timely localization of repair proteins to DNA substrates and epigenetic marks can modulate this critical decision.