Recombination is an important aspect of nucleic acid metabolism. Besides bringing about genetic exchanges, recombination appears to be required for the accurate segregation of chromosomes during meiosis and plays an important role in the repair of various classes of DNA damage. Aberrant recombination events are associated with genetic defects and different types of cancer. Tumor promoters like TPA stimulate recombination and cell lines. derived from' individuals with Blooms syndrome and other diseases like ataxia telangiectasia appear to have elevated levels of recombination. Understanding the biochemical mechanism(s) of recombination is central to our knowledge of these diseases. While some aspects of the mechanism(s) of recombination are becoming clear, the overall enzymatic mechanism(s) of recombination is poorly understood. The long term goal of this proposal is to determine the enzymatic mechanisms of recombination in eukaryotes using the yeast Saccharomyces cerevisiae as a model organism. Previous studies performed under the auspices of this project have led to the development and characterization of an in vitro system that uses extracts of mitotic cells to catalyze recombination events. Analysis of the in vitro recombination reaction has led to the purification of 4 proteins having properties consistent with their being involved in recombination. This analysis of recombination will be continued by following 7 lines of experimentation. 1) The biochemical properties of strand exchange protein 1 (SEP l) will be characterized using now available overproduced protein. 2) The properties conferred by sep1 mutations will be studied in greater detail to gain additional insights into the role SEP l plays in the cell. 3) SF1, an SEPI stimulatory factor that is also a strand exchange protein, will be overproduced and its biochemical properties determined in detail. 4) The effects of viable sfl insertion and deletion mutations on sporulation, recombination and repair will be determined. 5) The biochemical and genetic properties of intact heterotrimeric RPA, an SEP1 stimulatory factor, will be determined. 6) Additional proteins thought to be involved in catalyzing recombination will be purified and characterized. These include proteins such as endonucleases and exonucleases predicted by mechanistic studies of recombination and additional homologous pairing proteins, 3 additional already purified SEPI stimulatory factors and an already purified Holliday junction processing enzyme. And, 7) an already developed in vitro recombination system will be, used in conjunction with in vitro complementation and reconstitution approaches to identify additional recombination proteins. The ultimate goal of these studies will be to reconstitute recombination with purified proteins and defined DNA substrates and determine the mechanism(s) of these reactions.
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