Mechanism of Meiotic Recombination

Division Of Basic Sciences - Nci

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Abstract

Meiotic double-strand DNA break (DSB) repair by homologous recombination occurs via multiple processes defined by distinct decisions points. One important decision involves partner choice, between recombining with the sister chromatid (the dominant repair partner during mitosis) or with the homolog (the homologous chromosome of different parental origin, the preferred partner during meiosis). Another important decision involves recombination pathway choice, between producing crossovers, where flanking chromosome sequences are exchanged, or noncrossovers. A signature contribution of our group was the demonstration that crossover and noncrossover recombination proceed via different mechanisms that diverge after initial stages of strand invasion, and that feature different biochemical activities and genetic requirements. Work during previous review periods had shown that the conserved Sgs1-Top3-Rmi1 helicase-topoisomerase complex (STR) is responsible for partitioning early recombination events between noncrossover and crossover pathways. Sgs1-Top3-Rmi1 is the yeast homolog of the mammalian BLM helicase-Top3alpha-BLAP75 complex, implicated in cancer avoidance and recombination control in humans. We showed that all three members of the yeast complex are essential for normal recombination partner choice and for population of regulated meiotic crossover and noncrossover recombination pathways. Based on these findings, we hypothesized that STR, by promoting frequent disassembly of early strand invasion intermediates, acts as a chaperone for early recombination intermediates. We hypothesized that these repeated cycles of strand invasion and disassembly would result in template switching, which in turn would lead to recombinants with mosaic parental strand contributions. This hypothesis has now been confirmed by high-throughput sequencing of recombinants that occur in a highly polymorphic test interval; more than 2/3 of recombinants display clear evidence for template switching, multiple strand invasions, or both. In addition, we uncovered evidence for activities specific to the crossover pathway, including branch migration (2/3 of crossovers) and exonucleolytic gap-formation (1/3 of crossovers). Current work is aimed at determining the proteins responsible for these activities. Other work is aimed at confirming branch migration by mapping the location of Holliday junctions in recombination using a novel method we have developed to specifically purify Holliday junction-containing intermediates. Finally, we are studying how chromosome structure, specifically the meiotic chromosome axis, contributes to the regulation of recombination. A meiosis-specific subset of chromosome axis components, the Hop1 and Red1 proteins, are important for meiotic DSB formation and partner choice, and are enriched in some regions of the genome relative to others. Using a novel method to recruit axis proteins to regions that are normally depleted of these proteins, we have shown that high concentrations of the Hop1 protein are necessary and sufficient for meiotic DSB formation, but the recombination events initiated by these DSBs do not follow canonical meiotic recombination pathways. We are currently determining the mechanism by which Hop1 promotes DSB formation, and what additional factors are needed for Hop1-dependent DSBs to be repaired by canonical meiotic recombination mechanisms.