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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MBC-2 (01))
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Portnoy, Matthew
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University of Wisconsin Madison
Schools of Earth Sciences/Natur
United States
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Stanage, Tyler H; Page, Asher N; Cox, Michael M (2017) DNA flap creation by the RarA/MgsA protein of Escherichia coli. Nucleic Acids Res 45:2724-2735
Lewis, Jacob S; Spenkelink, Lisanne M; Jergic, Slobodan et al. (2017) Single-molecule visualization of fast polymerase turnover in the bacterial replisome. Elife 6:
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Bakhlanova, Irina V; Dudkina, Alexandra V; Wood, Elizabeth A et al. (2016) DNA Metabolism in Balance: Rapid Loss of a RecA-Based Hyperrec Phenotype. PLoS One 11:e0154137
Jaszczur, Malgorzata; Bertram, Jeffrey G; Robinson, Andrew et al. (2016) Mutations for Worse or Better: Low-Fidelity DNA Synthesis by SOS DNA Polymerase V Is a Tightly Regulated Double-Edged Sword. Biochemistry 55:2309-18
Ronayne, Erin A; Wan, Y C Serena; Boudreau, Beth A et al. (2016) P1 Ref Endonuclease: A Molecular Mechanism for Phage-Enhanced Antibiotic Lethality. PLoS Genet 12:e1005797
Leite, Wellington C; Galvão, Carolina W; Saab, Sérgio C et al. (2016) Structural and Functional Studies of H. seropedicae RecA Protein - Insights into the Polymerization of RecA Protein as Nucleoprotein Filament. PLoS One 11:e0159871
Kim, Taejin; Chitteni-Pattu, Sindhu; Cox, Benjamin L et al. (2015) Directed Evolution of RecA Variants with Enhanced Capacity for Conjugational Recombination. PLoS Genet 11:e1005278
Chen, Stefanie H; Byrne, Rose T; Wood, Elizabeth A et al. (2015) Escherichia coli radD?(yejH) gene: a novel function involved in radiation resistance and double-strand break repair. Mol Microbiol 95:754-68
Rajendram, Manohary; Zhang, Leili; Reynolds, Bradley J et al. (2015) Anionic Phospholipids Stabilize RecA Filament Bundles in Escherichia coli. Mol Cell 60:374-84

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