Abstract
Crossing over between homologous chromosomes occurs during the prophase of meiosis I and is critical for chromosome segregation. In baker’s yeast, two heterodimeric complexes, Msh4-Msh5 and Mlh1-Mlh3, act in meiosis to promote interference-dependent crossing over. Mlh1-Mlh3 also plays a role in DNA mismatch repair (MMR) by interacting with Msh2-Msh3 to repair insertion and deletion mutations. Mlh3 contains an ATP-binding domain that is highly conserved among MLH proteins. To explore roles for Mlh3 in meiosis and MMR, we performed a structure−function analysis of eight mlh3 ATPase mutants. In contrast to previous work, our data suggest that ATP hydrolysis by both Mlh1 and Mlh3 is important for both meiotic and MMR functions. In meiotic assays, these mutants showed a roughly linear relationship between spore viability and genetic map distance. To further understand the relationship between crossing over and meiotic viability, we analyzed crossing over on four chromosomes of varying lengths in mlh3Δ mms4Δ strains and observed strong decreases (6- to 17-fold) in crossing over in all intervals. Curiously, mlh3Δ mms4Δ double mutants displayed spore viability levels that were greater than observed in mms4Δ strains that show modest defects in crossing over. The viability in double mutants also appeared greater than would be expected for strains that show such severe defects in crossing over. Together, these observations provide insights for how Mlh1-Mlh3 acts in crossover resolution and MMR and for how chromosome segregation in Meiosis I can occur in the absence of crossing over.
Highlights
Crossing over between homologous chromosomes occurs during the prophase of meiosis I and is critical for chromosome segregation
Meiotic recombination in Saccharomyces cerevisiae is initiated by the induction of approximately 1402170 SPO11-dependent double-strand breaks (DSBs) that are located throughout the genome (Cao et al 1990; Gilbertson and Stahl 1996; Keeney et al 1997; Robine et al 2007; Chen et al 2008)
To further analyze the role of Mlh3 in meiosis, we analyzed crossing over on four chromosomes in mlh3Δ mms4Δ cells and observed a strong decrease in crossing over at all intervals, but higher spore viability than would be expected for strains that show such strong crossover defects. Together these observations provide insights for how Mlh1-Mlh3 acts in crossover resolution and mismatch repair (MMR), and for how chromosome segregation in Meiosis I can occur in the absence of crossing over
Summary
Media S. cerevisiae strains were grown at 30° in either yeast extract-peptone, 2% dextrose media, or minimal selective media (SC) containing 2% dextrose, sucrose, or galactose (Rose et al 1990). Plasmids used for the dominant-negative assay were constructed by QuickChange mutagenesis using pEAE220 (S288C, GAL10-MLH3, 2m, URA3) as a template (Nishant et al 2008). The SK1 mlh alleles described in this study were introduced by gene replacement into SK1 congenic and isogenic strain backgrounds (Tables 1 and 2). The isogenic SK1 strain EAY1062 [lys2::InsE-A14 (Nishant et al 2008)] was used to measure the effect of mlh mutations on mutation rate (Table 3). Lys+ reversion assays The mlh allele constructs were transformed into EAY2037 (SK1, mlh3Δ::KANMX4, lys2::InsE-A14), and strains were analyzed for reversion to Lys+ (Tran et al 1997). For the dominant-negative assays, EAY1269 bearing pEAE220 and mutant derivatives were grown for 5 d on uracil dropout SC agar plates containing 2% sucrose or 2% sucrose and 2% galactose. Using GAL10-MLH3 and mlh1Δ as controls, we analyzed 11 independent colonies from two independent transformations
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