During the past year, we have made progress in the following areas: Genome-wide mapping of DNA damage. Meiotic recombination is initiated by DNA double-strand breaks (DSBs), whose location and time of formation are tightly controlled. Our previous work developed a novel method to isolate intermediates in DSB repair, and applied this method to a genome-wide map of meiotic DSBs, based on microarray analysis, with a resolution of about 500 nucleotides. While this method had sufficient resolution to document overall DSB distributions, further mechanistic insight would be gained by knowing precise DSB locations. We therefore developed a method to determine the genome-wide location of meiotic DSBs at single-nucleotide resolution, using a modified high-throughput sequencing protocol. Our initial aim is to determine the substrate requirements of proteins that form DSBs, the factors that control DSB location, frequency, and timing, and how these factors change over evolutionary time. Future work will modify this approach to allow high-resolution, genome-wide maps of spontaneous DNA damage in growing cells, and DNA damage induced by carcinogenic and cancer chemotherapeutic agents. Partner choice during meiotic recombination. Recombination can occur between sister chromatids or between homologous chromosomes of differing parental origin, called homologs. Inter-sister recombination, which is the predominant form of recombination during the mitotic cell cycle, was thought to be greatly reduced during meiosis, since interhomolog recombination is required to pair homologous chromosomes and to ensure their disjunction at the first meiotic division. We have now show that inter-sister recombination occurs much more frequently that previously thought during meiosis, and that the increased level of interhomolog recombination that occurs during meiosis results from a modest (3-fold) reduction in the rate of intersister recombination. These findings are prompting a revision of the nature of recombination partner choice control, in particular the regulatory mechanisms that limit intersister and promote interhomolog recombination. Resolution of recombination intermediates during meiosis and the mitotic cell cycle. Our previous work has shown that double Holliday junction (dHJ) recombination intermediates, which form at high levels during meiosis, are mostly resolved as crossover products, in a reaction that is regulated by the polo-like kinase, Cdc5. Structure-specific endonucleases suggested as being responsible for dHJ intermediate resolution include the Mus81/Mms4, the Slx1/Slx4, and the Yen1 endonucleases. Using genetic and molecular approaches, we have now shown that most dHJ intermediates are resolved in the absence of all three of these endonucleases, suggesting that a fourth, yet unidentified nuclease is responsible for most meiotic recombination intermediate resolution. We also showed that Mus81/Mms4, Slx1/Slx4 and Yen1 are important for resolving the abnormal recombination intermediates that accumulate in the absence of the Sgs1 helicase (the budding yeast homolog of the helicase mutated in Bloom's syndrome), suggesting that these nucleases act to chaperone recombination intermediates that form outside of the normal crossover-directed meiotic recombination pathway. We have also taken advantage of a property of budding yeast meiosis, namely its reversibility, to examine mechanisms of recombination intermediates during the mitotic cell cycle. dHJ intermediates are accumulated in meiotic cells, which are then returned to mitotic growth conditions, where they rapidly exit meiosis and resume the mitotic cell cycle. In contrast to meiosis, where dHJ intermediates are resolved primarily as crossover recombinants, the same intermediates are rapidly resolved without crossover formation upon return to growth. We have identified the Sgs1 helicase as being required for this resolution;in the absence of Sgs1, dHJ intermediate resolution is delayed, and about half are resolved as crossovers. Current work is aimed at identifying the factors that catalyze and that regulate this resolution process.
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