The goal of this proposal is to determine the molecular mechanisms used by RecQ DNA helicases. RecQ involvement in DNA replication, recombination, and repair has been demonstrated in both bacteria and eukaryotes, but the precise roles played by this family of helicases remain unclear. Their importance, however, is unambiguous -- mutation of one of three human RecQ genes leads to genome instability and an elevated predisposition to cancer in Werner's, Bloom's, and Rothmund-Thompson syndromes as well as to premature aging in Werner's and Rothmund-Thompson syndromes. RecQ proteins, therefore, represent a paradigm linking genome stability to disease states as well as for understanding the connections between DNA replication, recombination, and repair. This makes a precise, molecular understanding of the mechanisms of the RecQ family important to both basic and medical science. The experiments described in this proposal aim to elucidate the biological functions of RecQ proteins by resolving their molecular mechanisms of action in vitro and in viva. The approach focuses biochemical, high-resolution structural, and genetic methods on bacterial RecQ proteins to delineate the structure-function relationship that supports activity in the enzyme. These experiments include: (1) a determination of the roles of the conserved domains within the RecQ family (helicase, RecQ-Ct, and HRDC domains), (2) elucidation of the quaternary and tertiary structures of RecQ, and (3) identification of RecQ-containing protein complexes in bacterial cells and a study of the effects of interacting proteins on RecQ activity in vitro. A multidisciplinary approach will allow biochemical links to be made between the cellular roles of RecQ proteins and their molecular mechanisms of action.
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