The studies described in this research proposal are directed at determining the biochemical mechanism and biological function of DNA helicases. The helicases to be investigated include the Escherichia coli recBCD, recQ, ruvAB, and uvrD proteins. These proteins play important roles in genetic recombination and DNA repair, yet the specific biochemical mechanism of their action is unclear. The studies of the multifunctional recBCD enzyme will focus on the nature of its interaction with the recombination hotspot sequence, chi. The chi sequence was recently found to be a regulatory sequence that attenuates the nuclease activity but not the helicase activity of recBCD enzyme. The experiments described in this proposal are designed to test a number of hypotheses regarding the biochemical nature of this interaction and to further refine our understanding of this novel phenomenon. The studies of the recQ, ruvAB, and uvrD proteins are designed to characterize the helicase and ATPase activities of these proteins. The biochemical mechanism of helicase action will be investigated by taking advantage of two different fluorescent helicase assays that were developed in my laboratory. These assays are rapid, continuous, and quantitative; consequently, they possess many advantages over traditional methods. The relationship of the ATPase activity of each helicase to its DNA unwinding activity will be examined. To better understand the physical basis of the DNA unwinding activity, the equilibrium DNA- and ATP-binding properties of these proteins will be examined. Finally, studies on these helicases will also include characterization of the biochemical properties of selected mutant DNA helicases in order to probe aspects of biochemical mechanism and to relate enzymatic activity with biological function.
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