We continued research on the molecular mechanism of meiotic recombination in the yeast Saccharomyces cerevisiae, with a focus on three aspects of this complex biological process. We examined the initiation event in meiotic recombination, double-strand DNA break formation, with the aim of identifying the enzyme(s) responsible for break formation, their substrate requirements, and the factors that control the location and frequency of breaks. These factors include aspects of both chromatin and higher-order chromosome structure. A particular focus has been the role of chromosome structural elements, specifically centromeres and telomeres, in repressing double-strand break formation. We are also examining the relationship between meiotic DNA replication and recombination, and have demonstrated a direct mechanistic link between these two processes. We developed techniques to isolate unstable intermediates in meiotic recombination and are currently analyzing their structure. These latter experiments have demonstrated that crossover and noncrossover-associated repair of meiotic double-strand breaks proceed by distinct mechanisms, rather than being produced by alternate resolution of a common recombination intermediate. A second, related project involves the use of DNA microarrays to study gene expression and chromosome structure during meiosis, using mRNA and chromatin immunoprecipitation analyses. We are currently using DNA microarrays containing fragments covering a yeast chromosome (chromosome III) to examine meiotic chromosome structure, and are constructing microarrays covering the entire yeast genome in collaboration with four other research groups at the NCI.
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