Biochemical Analysis of Genetic Recombination in Yeast

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DESCRIPTION (provided by applicant): The long-term objective of this research program is to understand the molecular mechanisms of homologous recombination in eukaryotes. Homologous recombination plays two essential roles during the life cycle of most organisms. It is required to repair lethal lesions in DNA, such as double-strand breaks, and it is essential for the pairing and segregation of homologous chromosomes during meiosis. The importance of these functions is evidenced by increased mutagenesis, and mitotic and meiotic aneuploidy in the absence of recombination. Since many human cancer-prone syndromes are associated with increased genome instability, an understanding of the mechanisms of recombination is likely to be important in understanding these diseases. Our goals are to identify the genes and proteins required for homologous recombination in the yeast, Saccharomyces cerevisiae. The resection of DNA ends to generate single-stranded DNA is an essential step in recombination. Mre11p is a nuclease and is required to process meiosis-specific double-strand breaks (DSBs), but the Mre11 nuclease activity is not required for processing HO-induced DSBs in mitotic cells. Nucleases that are redundant with Mre11 will be identified using genetic screens or by screening a library of yeast GSTfusion proteins. The Mre11 complex will be purified from mitotic and meiotic cells to identify novel associated factors with Mre11 and for biochemical characterization. We developed a colony color-sectoring assay to identify genes involved in mitotic recombination. Using this assay we characterized a RAD51-independent recombination pathway that requires the Rad52 homologue, Rad59. RAD51-independent recombination occurs by two major pathways, break-induced replication (BIR) and single-strand annealing (SSA). The role of RAD59 in BIR and oligonucleotide-directed gene targeting will be determined. Residues of Rad59 important for DNA repair and RAD51-independent recombination will be identified. Studies with the Rad52/Rad59 complex will address the role of the complex in strand annealing and strand invasion to prime DNA synthesis. Other genes that function in the RAD51 -independent recombination pathway will be identified using the colony-color sectoring assay and by interaction of their products with Rad59 in a two hybrid screen