BIOCHEMICAL ANALYSIS OF GENETIC RECOMBINATION IN YEAST

Columbia University Health Sciences, New York NY

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Abstract

The long term objectives are 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 genetic diseases are associated with increased genome instability, an understanding of the mechanisms of recombination is likely to be important in understanding these diseases. Furthermore, homologous recombination has practical application as a tool for gene therapy. Our goals are to identify the proteins that catalyze homologous recombination in the yeast Saccharomyces cerevisiae using both genetic and biochemical approaches. Yeast offers several advantages for these studies. It is a relatively simple, unicellular eukaryote that is easily grown mitotically in a haploid or diploid state, and that can be induced to undergo meiosis by growth in simple liquid medium. It has a well-defined genetic system that has enabled the isolation of a number of mutants altered in recombination. Finally, the complete DNA sequence of yeast has been determined. Most of the proposed research is focused on the use of a colony color sectoring assay to identify genes involved in mitotic recombination. Using this assay we have shown that mitotic recombination is reduced only 5-fold by mutation of RAD51, which encodes a homolog of bacterial RecA proteins. RAD59, which encodes a Rad52 homolog, was isolated by its requirement for RAD51-independent recombination. The Rad59 protein will be purified and characterized biochemically. Interaction with Rad52 will also be tested. Other genes that function in the RAD51-independent recombination pathway, in particular, genes involved in crossing over, will be identified using the colony sectoring assay. Finally, we plan to characterize the MRE11 gene. Genetic and molecular studies indicate that Mre11 is the prime candidate for an activity that processes the ends of double-strand breaks. Conserved residues in Mre11 predicted to be important for nuclease activity will be altered and tested for in vivo function, and the Mre11 protein complex will be purified to test the hypothesis that is a nuclease.