Crossovers generated during the repair of programmed meiotic double-strand breaks must be tightly regulated to promote accurate homolog segregation without deleterious outcomes, such as aneuploidy. The Mlh1–Mlh3 (MutLγ) endonuclease complex is critical for crossover resolution, which involves mechanistically unclear interplay between MutLγ and Exo1 and polo kinase Cdc5. Using budding yeast to gain temporal and genetic traction on crossover regulation, we find that MutLγ constitutively interacts with Exo1. Upon commitment to crossover repair, MutLγ–Exo1 associate with recombination intermediates, followed by direct Cdc5 recruitment that triggers MutLγ crossover activity. We propose that Exo1 serves as a central coordinator in this molecular interplay, providing a defined order of interaction that prevents deleterious, premature activation of crossovers. MutLγ associates at a lower frequency near centromeres, indicating that spatial regulation across chromosomal regions reduces risky crossover events. Our data elucidate the temporal and spatial control surrounding a constitutive, potentially harmful, nuclease. We also reveal a critical, noncatalytic role for Exo1, through noncanonical interaction with polo kinase. These mechanisms regulating meiotic crossovers may be conserved across species.
Meiotic recombination forms crossovers important for proper chromosome segregation and offspring viability. This complex process involves many proteins acting at each of the multiple steps of recombination. Recombination initiates by formation of DNA double-strand breaks (DSBs), which in the several species examined occur with high frequency at special sites (DSB hotspots). In Schizosaccharomyces pombe DSB hotspots are bound with high specificity and strongly activated by linear element (LinE) proteins Rec25, Rec27, and Mug20, which form co-localized nuclear foci with Rec10, essential for all DSB formation and recombination. Here, we test the hypothesis that Rec10's nuclear localization signal (NLS) is crucial for coordinated nuclear entry after forming a complex with other LinE proteins. In NLS mutants, all LinEs were abundant in the cytoplasm, not the nucleus; DSB-formation and recombination were much reduced but not eliminated. Nuclear entry of limited amounts of Rec10, apparently small enough for passive nuclear entry, can account for residual recombination. LinEs are related to synaptonemal complex proteins of other species, suggesting that they also share an NLS, not yet identified, and protein complex-formation before nuclear entry.
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