DNA helicases catalyze NTP hydrolysis-dependent unwinding of duplex DMAto provide single-stranded DNA (ssDNA) for use as a template or reaction intermediate in DNA transactions. We have focused our efforts on DNA helicases in E. coli and the budding yeast Saccharomyces cerevisiae. The long-range goal of this research program is to understand, in enzymatic and molecular terms, the mechanism of action and cellular role of several important DNA helicases in E. coli and yeast. Our focus during the next grant period will be on DNA helicases II and IV from E. coli, and Sgslp and Hmilp from yeast. The 1st and 2nd aims will continue our efforts to define the interaction between helicase II (UvrD) and MutL, two proteins that play a critical role in maintaining genomic stability. We will determine the mechanism by which MutL stimulates UvrD-catalyzed unwinding using biochemical approaches. In addition, we will evaluate, through genetic studies, the importance of the interaction between these proteins by identifying mutants that fail to interact. The 3rd aim proposes to identify proteins that interact with and modulate the activity of UvrD and helicase IV using a novel biochemical approach. We will biotinylate the target proteins in vivo and identify interacting proteins in pull-down assays from cell extracts. The effect of these proteins on the biochemical activities of each helicase will be evaluated in biochemical and genetic experiments. This will shed additional light on the roles these proteins play in the cell. The final two aims focus on two DNA helicases from budding yeast. The Sgslp has been expressed as a full length protein in baculovirus and will be thoroughly characterized as a helicase with regard to unwinding mechanism (processive vs. distributive), substrate preferences (DNA vs. RNA vs. DNA-RNA) and substrate structure. In addition, the interaction between Sgslp and Topoisomerase III will be investigated using biochemical methods. Preliminary studies suggest that topo III modulates the biochemical activity of Sgslp. These studies will provide additional information relevant to the role of Sgslp in maintaining genomic stability. Finally, a mitochondrial helicase, Hmilp, has been identified and partially characterized in genetic studies. A thorough biochemical description of this protein is lacking and will shed light on the role this protein plays in mtDNA metabolism.This protein will be expressed and fully characterized in biochemical assays.
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