The goal of this proposed research is to determine the molecular mechanisms by which enzymes utilize the free energy of ATP hydrolysis to bring about changes in the structure of the DNA helix. The research will focus on the DNA pairing and unpairing reactions that occur during homologous genetic recombination. The mechanisms of action of two proteins from E. coli will be analyzed: the recA protein and the uvrD protein. The recA protein is responsible for the pairing of DNA molecules in new hybrid combinations during homologous recombination. In this research, several new DNA pairing reactions that are designed to elucidate the individual steps that comprise a strand invasion event will be investigated. The general mechanism of the recA protein-promoted homologous alignment of DNA chains will be determined and the role of ATP hydrolysis in the process will be defined. In a second project, the molecular mechanism of the ATP-dependent, DNA helicase-catalyzed unwinding of duplex DNA will be studied. The research will initially focus on the uvrD protein, which has been implicated in DNA recombination and repair. The methodology that is developed will be applicable to the mechanistic study of DNA helicases in general. A careful study of the mechanisms of action of individual enzymes will provide a basis for studying the coordinate action of multiprotein systems during the course of a complete recombination event.
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