The long-term objective of this research proposal is to contribute to the biochemical understanding of the mechanism of homologous recombination. The general approach is to enzymatically characterize the purified proteins known to participate in genetic recombination in E. coli and to determine their enzymatic mechanism when acting individually or as component of a more complex system. These proteins include the recA, SSB, and recBCD proteins of E. coli. The specific approach is to pursue physical-biochemical, enzymatic, and structure-functional characterization of a protein that plays a central role in genetic recombination, the recA protein of E. coli. To assess the biological significance of the many recA protein activities and to determine how they contribute to enzymatic function, the biochemical properties of mutant recA proteins will be examined. Finally, in order to better understand how the activities of the recA protein are coordinated with those of SSB and recBCD enzymes to promote the homologous pairing, in vitro assays that require the activities of these proteins will be examined mechanistically. Understanding the molecular mechanism of genetic recombination should shed light aberrant on biological events such as chromosomal translocations and error-prone repair and should contribute to the fundamental biochemical knowledge required to develop useful gene replacement therapies based on homologous recombination.
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