The broad objective of this proposal is to understand the biochemical mechanism of the steps that comprise the process of homologous recombination. Genetic recombination is a fundamental biological process that involves the processing of broken DNA, the homologous recognition and exchange of DNA strands between participants, and the resolution of the recombination intermediates. It is an important cellular process that is used by all organisms to repair DNA damage, restart DNA replication, and generate genetic diversity. In E. Coli, several dozen enzymes are involved in recombination. Two broad objectives are planned. The first is to reconstitute genetic recombination in vitro. The second is to understand the biochemical mechanism by which the component proteins of this process function. The research objectives will be achieved by reconstructing various biochemical steps of the recombination process in vitro, using purified components from E. coli. The experiments will include most of the enzymes of recombination, including RecA, SSB, RecBCD, RecQ, RecJ, RecF, RecO, RecR, RuvA, RuvB, and RuvC proteins, as well as the DNA polymerases and topoisomerases that are important to the recombination process. The mechanistic studies will focus on the substrate range and reaction specificity of several novel DNA and RNA strand exchange reactions, structural and functional aspects of these reactions, and visualization of these processes at the single-molecule level. These studies will reveal how the steps that comprise genetic recombination occur at the molecular level, and they will provide a biochemical understanding of these steps. Knowledge of the mechanism of recombination should provide insight into the manner by which DNA breaks are repaired, how aberrant chromosomal translocations occur, and passable new experimental approaches for gene replacement therapies.
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