Studies in the model eukaryote Saccharomyces cerevisiae have revealed that homologous recombination (HR) provides a major mechanism for eliminating DNA double-stranded breaks (DSBs) induced by ionizing radiation or are associated with injured DNA replication forks. During the repair process, the ends of the DNA breaks are resected nucleolytically to yield 3' ssDNA tails, which are bound by HR factors. The nucleoprotein complex thus formed then conducts a search to locate an undamaged DNA homolog, and catalyzes the formation of a DNA joint, called D-loop, with the homolog. Resolution of the D-loop can proceed via at least three mechanistically distinct pathways, two of which generate only non-crossover recombinants and are therefore more adept at genome preservation, with the remaining pathway being able to produce crossovers frequently. Proteins encoded by evolutionarily conserved genes of the RAD52 epistasis group catalyze the HR reaction. Our studies have provided insights into the mechanistic underpinnings of the HR machinery that harbors proteins of this gene group. In this renewal project, a combination of biochemical, genetic, and other cell-based approaches will be employed to (i) define the mechanism of action of the DNA motor-driven path of DSB end resection and its functional crosstalk with Exo1-mediated resection in both yeast and human cells, and (ii) delineate the roles that the conserved Rad51 paralogs fulfill in the assembly of Rad51-ssDNA nucleoprotein filaments. The results from our endeavors will allow us to formulate detailed models to elucidate HR mechanisms in eukaryotes. Given the importance of HR-mediated chromosome damage repair in tumor suppression, our work also has direct, strong relevance to cancer biology.
Cancers are characterized by genomic instability and rearrangements, which can be induced by exposure of cells to DNA damaging agents such as ionizing radiation or are associated with the stalling or collapse of DNA replication forks. The proposed studies will continue to delineate the mechanism by which eukaryotic cells avoid deleterious genome rearrangements via the process of homologous recombination. The results from our research endeavors have direct relevance to radiation and cancer biology.
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