BIOCHEMICAL ANALYSIS OF GENETIC RECOMBINATION IN YEAST

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Our long term objectives are to understand the molecular mechanisms of homologous recombination in eukaryotes. Homologous recombination plays two important, but seemingly contradictory roles in the life cycle of most organisms. On one hand it is important to generate diversity by creating new combination of genes, or parts of genes. On the other hand it is required for the faithful repair of DNA lesions in mitotic cells, and for segregation of chromosomes during meiosis. The importance of the latter functions is evidenced by increased mutagenesis, and mitotic and meiotic aneuploidy in the absence of recombination functions. 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. We have chosen to use the yeast Saccharomyces cerevisiae as a model system for these studies since it is easily manipulated genetically and biochemically and, in addition, shows high frequency mitotic and meiotic recombination. The primary goal of this proposal is to purify proteins that catalyze recombination and to identify the genes that encode them. Evidence has accumulated that 5'-3' exonuclease activity is required to process double-strand break sites prior to strand invasion. We have identified several 5'-3' exonuclease activities in mitotic extracts. We plan to purify one of these and to use the purified protein to identify the gene encoding the nuclease. Mutants will then be constructed to determine its role in repair and recombination. We have established a procedure for preparing figure-8 DNA molecules that contain a Holliday junction. Using such DNA as a substrate we have identified an activity that resolves Holliday junctions. This activity will be purified and its cellular function determined using reverse genetics. The figure-8 substrate will also be used to identify and purify branch migration promoting activities. In addition to these biochemical studies, we plan to utilize a sensitive colony sectoring screen to isolate mutants that decrease spontaneous mitotic recombination. Our goal in the isolation of more mutants is to better understand the genetic control of recombination, and to increase the available pool for screening for defects in biochemical activities.