BIOCHEMICAL MECHANISM AND FUNCTION OF DNA HELICASES

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DNA helicases represent a ubiquitous class of nucleic acid enzymes. These proteins "unwind" (i.e., denature) dsDNA into separate single- strands of DNA, in a reaction requiring nucleoside triphosphate hydrolysis. The broad objective of this proposal is to understand the biochemical mechanism and biological function of DNA helicase action, i.e., how helicases translocate along single-stranded DNA (ssDNA) to unwind double-stranded (dsdna) and, having done so, how this effort is translated into biological function (i.e., useful work). An understanding of these fundamental questions will contribute to our understanding of the function of these helicases and of translocating proteins, in general. We plan to study two DNA helicases. The RecBCD and RecQ enzymes of E. coli. The RecBCD enzyme is a multi-subunit, multifunctional enzyme that possesses both helicase and nuclease activities; it also has the ability to recognize, while translocating, a specific DNA sequence called khi, that regulates its nucleolytic activities. It is hoped that a detailed biochemical comparison of these helicases will help clarify the biochemical mechanism and biological function of DNA helicases. The long-term, specific aims of this proposal are to continue our investigations into the DNA helicase and nuclease activities of the RecBCD enzyme, and into the manner by which these activities are affected by interaction with the recombination hotspot sequence, khi. Furthermore, we wish to compare these findings to the behavior of the RecQ protein. This knowledge will contribute a general appreciation of the molecular events responsible for aberrant cellular processes. Mutations in genes that encode putative helicases are associated with human disorders as diverse as xeroderma pigmentosum (ERCC2 and ERCC3); Cockayne's syndrome (EERCC6); Bloom's syndrome (BLM, which is a RecQ homologue); and Werner's syndrome (WRN, which is also a RecQ homologue), showing that an understanding of these proteins is crucial for the understanding of disease processes as diverse as cancer and premature aging.