BIOCHEMICAL CHARACTERIZATION OF YEAST DNA HELICASES

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DESCRIPTION (Adapted from the applicant's abstract): The double stranded structure of DNA mandates the existence of a mechanism for unwinding the helix to expose single-stranded DNA (ssDNA) for use as a template or reaction intermediate in DNA replication, repair, recombination and perhaps transcription. The DNA helicases are enzymes which provide such a mechanism. These enzymes translocate through duplex DNA disrupting the hydrogen bonds that hold the two strands together in a reaction utilizing energy provided by NTP hydrolysis. More than ten DNA helicases have been described in the prokaryote E. coli; the role of each in DNA metabolism is currently being elucidated using a combination of biochemical and genetic studies. To date comparable detailed studies of DNA helicases in eukaryotic organisms have lagged behind. Recently, the principal investigator initiated a project aimed toward identifying and characterizing DNA helicases from the budding yeast Saccharomyces cerevisiae. Yeast has been chosen as an example of a eukaryotic organism that is amenable to both biochemical and genetic studies. Four previously undescribed DNA helicases have been identified by chemical criteria; three have been purified to apparent homogeneity. The long range goal of this project is to characterize, biochemically and genetically, the four new yeast DNA helicases that have been isolated. Each of these enzymes will be purified to homogeneity and characterized with respect to DNA/RNA substrate requirements, reaction mechanism (processive versus distributive), and interactions with other proteins. Enough protein will be purified to obtain sufficient amino acid sequence data to allow cloning of the gene encoding each enzyme. This will permit a genetic analysis to begin to elucidate the precise biochemical function of each of these helicases in the cell.