The RecA protein of E. coli promotes a DNA strand exchange reaction in vitro that provides a convenient molecular model for the central steps of homologous genetic recombination. This protein is the prototype for a family of recombinases found in all organisms. In humans, the primary RecA homologue (hRad51) is part of a major pathway for tumor suppression. The long-range goal of the research in this proposal is a detailed understanding of RecA-mediated DNA strand exchange. The hypothesis that recombinational DNA repair of stalled replication forks is the primary function of RecA protein provides an intellectual framework. The project has evolved to include the RecA proteins of E. coli and D. radiodurans, as well as the Rad51 protein of yeast. There are two major areas of emphasis: (a) structure-function relationships and (b) general biochemistry of RecA family recombinases. Structure-function efforts are focused on two initiatives. First, the C-terminus of RecA protein is a flap that modulates every RecA activity. The 25 amino acid residues at the C-terminus affect the pH-reaction profile of DNA strand exchange, mediate a Mg ion-dependent conformation change that activates RecA, inhibit the displacement of SSB from ssDNA, are responsible for inhibition by PEP, and regulate the RecA coprotease and SOS mutagenesis functions of RecA. All of these activities are under investigation. We also have several projects to elucidate the structure of RecA protein bound to DNA. Biochemical studies are focused on recombinase filament assembly and disassembly, DNA pairing and strand exchange, and the role of ATP hydrolysis in recombinase reactions. The filament studies include a search for mutants with enhanced DNA binding properties, an investigation of the path by which DrRecA filaments assembly on dsDNA, and a look at properties of Rad51 filament formation. An unusual priming effect of gap junctions on DNA strand exchange will be explored to provide new insight into DNA pairing processes. Finally, the hypothesis that RecA is a motor protein will be tested.
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