The goal of this proposal is to determine the enzymatic mechanisms of genetic recombination and repair of mismatched nucleotides in the yeast Saccharomyces cerevisiae. The basic approach that is being followed is to develop in vitro systems and biochemical assays that measure genetic recombination and mismatch repair in order to identify the biochemical intermediates and purify the proteins that are involved in genetic recombination and mismatch repair. Plasmid DNA and M13 phage DNA substrates that allow biochemical and genetic detection of homology dependent recombination events and the repair of mismatched nucleotides have been constructed and used to detect recombination and mismatch repair catalyzed by cell-free extracts of mitotic cells. The intermediates and products that are formed in these reactions will be characterized in detail to provide insight into the mechanisms of these reactions. Recombination substrates treated with mutagens, carcinogens, Gamma-irradiation and UV light will be constructed to access the effect of DNA damage on recombination in vitro. The effect of chromatin structure and the presence of recombination hotspots on recombination in vitro will be determined and a comprehensive study of the specificity of mismatch repair in vitro will be carried out. Cell-free recombination systems using extracts prepared form either meiotic cells or Gamma-irradiation or mutagen-induced cells will be developed and characterized. The proteins required for recombination and mismatch repair will be purified using a combination of in vitro complementation and reconstitution assays and characterized in detail. Studies on the purification and characterization of a S. cerevisiae Holliday Junction specific endonuclease will continue. The genes encoding proteins required for recombination and mismatch repair will be cloned in order to overproduce their gene products and to facilitate genetic analysis of the proteins to determine if they are required for recombination and mismatch repair in vivo. The ultimate goal of these studies will be to reconstitute recombination and mismatch repair reactions with purified proteins and determine the enzymatic mechanisms of these reactions.
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