MECHANISMS OF DOUBLE STRAND BREAK REPAIR IN YEAST

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The purpose of the proposed research is to determine the functions of the yeast DNA repair proteins Rad55 and Rad57. These two proteins were recently identified as RecA homologues suggesting a direct role in DNA repair and recombination. The RAD55 and RAD57 genes are required for the repair of double-strand breaks, for spontaneous mitotic recombination and for meiosis. The lack of functions required for double-strand break repair has deleterious effects, such as elevated rates of mutagenesis and chromosome loss, and meiotic lethality. As cancer susceptibility has been associated with defects in DNA repair functions it is possible that defects in the double-strand break repair pathway in mammals will also be involved in cancer progression. Furthermore, as the RAD genes are required for gene targeting in yeast, an understanding of their role in yeast may lead to the development of improved strategies for gene targeting in mammalian cells. The focus of this proposal it to determine the biochemical functions of Rad55 and Rad57 and their interactions with other Rad proteins in particular Rad51. Rad55 and Rad57 purified from yeast exhibit ATP-stimulated binding to single-stranded DNA, but no significant binding to duplex DNA. The purified proteins will be tested in other biochemical assays for RecA-like activities, including reannealing of complementary single-stranded DNA, strand exchange, D-loop formation, pairing of circular molecules and branch migration of Holliday junctions. As rad55 and rad57 mutants have similar phenotypes, and the proteins interact by the two hybrid system the two purified proteins will be mixed to determine whether the complex has altered or increased activity compared with the individual proteins. The two proteins will also be tested in combined reactions with Rad51 to determine whether they act as stimulatory factors for the Rad51-mediated strand-exchange reaction. Complex formation between Rad55 and Rad57 with other Rad proteins, or novel proteins, will be determined using affinity chromatography, immunoprecipitation and gel filtration chromatography. We also plan to investigate the role of RAD genes in plasmid gap repair. Experiments are designed to test our hypothesis that the Rad51 pathway is involved in DNA repair events that are resolved as non-crossovers. The effect of donor sequence location either plasmid or chromosomal on gap repair in rad strains will also be investigated. Finally, we plan to screen for hyperactive alleles of RAD51 that suppress the DNA repair defect of rad55/57 mutants. Characterization of such alleles may provide insight into the function of these proteins.