Joint Molecule Resolution During Meiotic Recombination

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PROJECT SUMMARY/ABSTRACT Crossing over facilitates accurate chromosome segregation during meiosis, the specialized cell division that produces gametes. A fundamental gap in our understanding of crossing over is how double-Holliday junction (dHJ) intermediates are specifically resolved into crossovers and how this process is regulated. This knowledge gap has persisted because of challenges to study dHJ resolution at the biochemical and cellular levels. These impediments are overcome in this proposal through breakthroughs in understanding the mechanism of dHJ resolution and the development of biochemical, genetic, and molecular reagents and approaches to study dHJ resolution in vitro and in vivo. These include the realization that dHJ resolution occurs through a mechanism that resembles the initiation steps of DNA mismatch repair and involves the nicking of dHJs by the MutLg endonuclease, trigged by the replicative clamp PCNA. These observations evoke a coherent model for crossover-specific dHJ resolution in which orientation-specific loading of PCNA targets MutLγ to incise specific DNA strands on both sides of the Holliday junctions (HJs). Resolution then occurs via migration of the HJs across the nicks driven by the Bloom helicase complex (BLM-TOP3-RMI1/2). Critically, the asymmetric pattern of nicks always yields a crossover. The long-term objective of this project, to understand the mechanism and regulation of crossover-specific dHJ resolution during meiosis, will be pursued through three aims. Aim 1 will test the key tenets of the crossover-specific dHJ resolution model in vitro by reconstituting the reaction using purified human proteins and a variety of DNA substrates containing HJs and loading sites for PCNA. In Aim2, predictions of the model will also be tested in vivo using the unrivaled suite of molecular-genetics tools available in budding yeast, augmented by new approaches to isolate the dHJ resolution step and study pertinent factors using real-time genetics. This aim will detect MutLγ-catalyzed nicks at prospective crossover sites and in dHJs poised for resolution; detect immature resolution products containing single-stranded flaps and gaps; confirm the crossover resolution role of Sgs1-Top3-Rmi1 (ortholog of the human BLM complex); determine the influence of MutLγ on alternative resolution pathways; and test the idea that Exo1 functions at crossover sites to stabilize MutLγ-catalyzed nicks. Aim 3 will exploit new insights into the regulation of dHJ resolution by polo-like kinase, Cdc5. Candidate targets identified by phospho- proteomics will be analyzed using molecular-genetics tools in budding yeast to understand how Cdc5 triggers dHJ resolution. The results of these aims will provide unprecedented insight into the mechanism and regulation of crossing over during meiosis. These findings will be germane to understanding pathologies associated with human meiosis, including infertility, miscarriage, congenital disease, and premature ovarian insufficiency, and are expected to define paradigms that are broadly relevant for chromosome biology.