Central to the progress of molecular biology and its application to human disease is a description of the molecular mechanisms of homologous recombination and the related repair reactions. Many recent advances in diagnosing and treating cancer and infectious diseases have come from studies of DNA metabolism, and gene therapy in humans cannot be fully realized without a detailed understanding of homologous recombination. Three long term goals of this continuing study are: 1) develop ways of utilizing electron microscopy (EM) to visualize nucleic acid-protein complexes; 2) apply this technology and biochemical assays to probe the molecular mechanisms of DNA strand exchange reactions catalyzed by RecA and UvsX proteins; 3) apply this approach to elucidate the reactions catalyzed by Human and other eukaryotic strand exchange proteins. A new technique for preparing DNA-protein complexes for EM termed fast-freezing avoids chemical fixation and air drying. This method will be refined and applied together with filter binding assays, ATPase assays, and gels assays to further investigate the homologous recombination reactions catalyzed by RecA and UvsX proteins. The structure of DNA in synaptic joints created by RecA and UvsX proteins will be examined land related to the properties of other 3 stranded DNAs. Mutants of RecA and non-hydrolyzable ATP analogs will be employed to address the role of ATP hydrolysis in strand exchange. The link between recombination and transcription will be probed using EM to visualize the relation between stationary or moving RNA polymerase molecules and the position of synaptic joints on those same DNAs. We will determine whether transcription can increase the rate of strand transfer and whether dual opposite transcription can enhance synapsis. In collaborative studies we will utilize EM to describe the structures and molecular mechanics of the reactions catalyzed by Human, Yeast, and Drosophila strand transferases. A novel drug column will be used to search for new DNA recombination and repair proteins from eukaryotic cells.
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