Competing Crossover Pathways Act During Meiosis in <em>Saccharomyces cerevisiae</em>

Abstract

IN most eukaryotic organisms the correct segregation of chromosomes at the first meiotic division requires reciprocal exchange between homologs. The physical manifestations of these crossover events, chiasmata, provide the contacts between homologous chromosomes that are necessary for segregation (Jones 1987). This cohesion or “chiasma binder” function ensures the generation of a bipolar spindle in which tension is generated at the kinetochores (Maguire 1974). The subsequent “programmed release of sister connections” is thought to be critical for meiosis I segregation (Storlazzi et al. 2003). Because of their importance, crossover events are highly regulated both within and among chromosomes. This regulation is clearly seen in Saccharomyces cerevisiae where two crossovers rarely occur within the same genetic interval (positive interference) and smaller chromosomes tend to display less positive interference than larger ones (Mortimer and Fogel 1974; Kaback et al. 1999). A net result of this regulation is that every chromosome, regardless of size, receives at least one reciprocal exchange (Jones 1987).

How are crossover events generated? Genetic and physical analyses of meiosis in S. cerevisiae showed that meiotic recombination is initiated by double-strand breaks that occur at specific chromosomal positions (reviewed in Keeney 2001). The repair of these breaks, preferentially using an unbroken homolog as a template, results in both reciprocal exchanges, termed crossovers (CO), and nonreciprocal exchanges, termed noncrossovers (NCO). The classical double-strand break repair (DSBR) model proposes that these events result from alternative resolutions of a common Holliday junction intermediate (reviewed in Pâques and Haber 1999). Recent studies, however, have suggested that COs and NCOs are processed via separate pathways. In support of this idea, meiotic mutants have been identified that specifically reduce the number of COs or allow NCO formation in the absence of COs (Ross-Macdonald and Roeder 1994; Sym and Roeder 1994; Hollingsworth et al. 1995; Storlazzi et al. 1995; Hunter and Borts 1997; Chua and Roeder 1998; Nakagawa and Ogawa 1999; Agarwal and Roeder 2000; Allers and Lichten 2001a,b; Hunter and Kleckner 2001; reviewed in Bishop and Zickler 2004; Hollingsworth and Brill 2004). Furthermore, the configuration of heteroduplex DNA seen in NCOs does not fit that predicted by the DSBR model (Porter et al. 1993; Gilbertson and Stahl 1996; Merker et al. 2003). Finally, the majority of Holliday junctions detected by physical analyses of cells induced for meiosis are processed into COs (Allers and Lichten 2001a,b; Börner et al. 2004).

In the budding yeast S. cerevisiae, the MER3, EXO1, MSH4, MSH5, MLH1, MLH3, MMS4, and MUS81 genes are each required to achieve wild-type levels of meiotic crossing over (Ross-Macdonald and Roeder 1994; Hollingsworth et al. 1995; Hunter and Borts 1997; Nakagawa and Ogawa 1999; Wang et al. 1999; Khazanehdari and Borts 2000; Borts et al. 2000; Tsubouchi and Ogawa 2000; delos Santos et al. 2001, 2003; Börner et al. 2004; Mazina et al. 2004). In each of these mutants, crossing over, as measured at specific genetic intervals, is reduced by less than threefold. The proteins encoded by these genes are thought to participate in the biochemical steps that lead to meiotic recombination. EXO1 is a 5′–3′ exonuclease that can act on duplex DNA ends (Tsubouchi and Ogawa 2000), MER3 is a meiosis-specific 3′–5′ helicase that is thought to process double-strand breaks into Holliday junction intermediates that form COs (Nakagawa and Ogawa 1999; Nakagawa and Kolodner 2002a,b; Mazina et al. 2004), and MUS81-MMS4 is an endonuclease that appears to preferentially cleave D-loops and half-Holliday junctions (Kaliraman et al. 2001; reviewed in Hollingsworth and Brill 2004). How these biochemical activities converge to regulate crossing over and interference remains a major question in the field.

Little is known about the roles of MSH4, MSH5, MLH1, and MLH3 in meiotic crossing over. Biochemical and genetic studies, however, have shown that they act in MLH1-MLH3 and MSH4-MSH5 complexes (Pochart et al. 1997; Wang et al. 1999; Wang and Kung 2002). While both MSH4 and MSH5 are homologs of the bacterial MutS mismatch repair protein, they do not appear to play a role in eukaryotic mismatch repair (Ross-Macdonald and Roeder 1994; Hollingsworth et al. 1995). In S. cerevisiae, msh4Δ and msh5Δ mutants display a two- to threefold reduction in crossing over, an increase in meiosis I nondisjunction, the loss of interference, and a subsequent loss in spore viability (Ross-Macdonald and Roeder 1994; Hollingsworth et al. 1995; Novak et al. 2001). In Caenorhabditis elegans, deletion of either the MSH4 or the MSH5 homolog results in a complete loss of crossing over that is accompanied by meiotic inviability (Zalevskyet al. 1999; Kelly et al. 2000). These observations have led to models in which MSH4-MSH5 acts to stabilize and/or resolve Holliday junction intermediates (Ross-Macdonald and Roeder 1994; Hollingsworth et al. 1995; Pochart et al. 1997). While meiotic crossover defects in mlh1Δ and mlh3Δ mutants appear less severe than those in msh4Δ and msh5Δ mutants, these mutants still display relatively high levels of meiosis I nondisjunction (Hunter and Borts 1997; Wang et al. 1999; Argueso et al. 2003). In contrast to msh4Δ strains, interference appears intact in mlh1Δ mutants (Argueso et al. 2003). Mlh1 ^and Mlh3 ^{mutant mice show severe defects in crossing over, resulting in sterility (}Edelmann et al. 1996; Woods et al. 1999; Lipkin et al. 2002). These results, in conjunction with epistasis and cell biological analyses in yeast and mice, suggest that MSH4-MSH5 and MLH1-MLH3 act in a common crossover pathway, with MSH4-MSH5 functioning prior to MLH1-MLH3 (this study; Hunter and Borts 1997; Wang et al. 1999; Santucci-Darmanin et al. 2000; Moens et al. 2002; Wang and Kung 2002).

The genetic, cytological, and biochemical studies summarized above suggest that crossing over in mice and C. elegans occurs primarily through an interference-dependent (MSH4-MSH5, MLH1-MLH3) pathway. Crossing over in S. cerevisiae, however, is thought to be controlled by both interference-dependent and interference-independent (MUS81-MMS4) mechanisms (Zalevsky et al. 1999; Khazanehdari and Borts 2000; delos Santos et al. 2001, 2003). The above observations, which suggest that organisms utilize interference-dependent and -independent crossover pathways to varying degrees, are supported by the following:

1. Mouse and C. elegans mutants defective in MSH4-MSH5 and MLH1-MLH3 complexes display severe crossover defects relative to the equivalent S. cerevisiae mutants (Edelmann et al. 1999; Woods et al. 1999; Zalevsky et al. 1999).

2. Crossing over, while reduced in S. cerevisiae mus81Δ and mms4Δ strains, is still subject to interference (delos Santos et al. 2001, 2003).

3. Crossing over and spore viability in S. cerevisiae msh5Δ mus81Δ or msh5Δ mms4Δ double mutants is significantly lower (approximately fivefold) than that in the single mutants (delos Santos et al. 2001, 2003; this study).

4. Schizosaccharomyces pombe mus81Δ strains display severe defects in spore viability and crossing over that can be explained by the lack of an interference-dependent pathway in this organism (Egel 1995; reviewed in Hollingsworth and Brill 2004).

To gain a better understanding of the relationships between members of different crossover pathways as well as the contribution of distributive pairing to the meiosis I division, we analyzed the effect of msh5Δ, mlh1Δ, and mms4Δ single, double, and triple mutations on meiotic crossing over at four consecutive genetic intervals on chromosome XV. Data from tetrad dissection and single spores were analyzed using newly developed software. Our data suggest that meiotic crossing over in yeast can occur through three distinct crossover pathways: MUS81-MMS4 promotes interference-independent crossing over in one pathway while both MSH4-MSH5 and MLH1-MLH3 participate in a second interference-dependent pathway (Argueso et al. 2003; delos Santos et al. 2003). MSH4-MSH5 appears to repress a third pathway that yields deleterious crossovers.

All mutants are isogenic derivatives of EAY1108/EAY1112 (materialsandmethods).

Rf refers to the recombination frequency in single spores determined by parental/(parental + recombinant) and cM indicates the genetic distance in tetrads calculated using the formula of Perkins (1949): 50 × {TT + (6 × NPD)}/(PD + TT + NPD).

Aberrant events were identified from the wild type, mms4Δ, mlh1Δ, and msh5Δ tetrad data presented in Tables 1 and ​and2.2. For the entire data set, 97% of the aberrant events were 3:1 or 1:3 tetrads; the rest were 4:0 or 0:4 tetrads. No postmeiotic segregation events were detected.

Interference was calculated from data presented in Tables 1 and ​and2.2. Asterisks indicate that the observed number of NPDs or COCs deviated significantly from the expected number based on a two-tail binomial test (Categorical Statistics Package, http://engels.genetics.wisc.edu), suggesting that interference is present in the interval (*P < 0.05; **P < 0.01).

Tetrads displaying the tetratype class at both the URA3-LYS2 and the LYS2-HIS3 intervals were examined for chromatid interference. P-values derived from χ ^{analysis indicate the probability that the number of tetrads with exchanges involving two, three, and four chromatids follows a 1:2:1 neutral distribution of double crossovers.} P-values <0.05 indicate a deviation from neutrality.

Acknowledgments