I propose to use plasmid DNAs to study the enzymatic and molecular mechanisms of genetic recombination in Sacharomyces cerevisiae (Yeast). Plasmid DNAs which can replicate in E. coli and yeast and undergo homologous recombination events in yeast will be constructed. These plasmids will be used to measure the in vivo rate of plasmid recombination in mitotic cells using physical assays in which the recombination event will generate a new restriction endonuclease fragment or new oligomeric form or a genetic assay in which the recombination event will regenerate an active gene. The substrates will also be used to determine the mechanism of plasmid recombination. The effect of chromosome structure on recombination will be tested by integrating the plasmid recombination substrates into yeast chromosomes. The effect of treatments which induce recombination in yeast, like induction of meiosis and treatment with mutagens, Gamma-irradiation and UV light will be studied using the plasmid substrates. The effect of yeast recombination deficient mutations and radiation sensitive mutations will also be examined. Segments of yeast DNA from chromosomal regions having high recombination rates will be inserted into the plasmid substrates and their effect on recombination rates will be measured to investigate the possible existence of recombination hotspots in yeast DNA. When plasmid recombination in vivo has been characterized, the plasmid recombination substrates will be used to investigate recombination in yeast extracts. Initial studies will concentrate on an in vitro recombination system which we have developed which catalyzes a recombination event between linear and circular plasmid DNA molecules and requires the product of the Rad52 gene. When recombination in yeast extracts has been optimized, the structure of the recombination products will be studied by electron microscopy. Proteins involved in yeast recombination will be purified using in vitro complementation and reconstitution assays. The ultimate goal of these studies will be to reconstitute a yeast recombination pathway from purified proteins and to study the enzymatic mechanism(s) of the pathway in detail.
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