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 recombinational DNA repair and homologous genetic recombination. 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 is the primary function of the RecA protein provides an intellectual framework. Beginning with this perspective, a set of experiments is set up to investigate two major issues: a) the existence and function of a putative triplex DNA pairing intermediate, and b) the role of ATP hydrolysis in RecA- mediated DNA strand exchange. Indirect evidence for a triplex DNA intermediate has been advanced by several laboratories over the last 5 years, but the existence of the triplex has remained controversial. Many of the outstanding objectives can be addressed definitively with experiments to probe the structure of a series of annealed oligonucleotides. This issue warrants attention because the proposed triplex DNA structure is novel, and because it provides a potential solution to the classic mechanistic problem of aligning two homologous DNAs for recombination. Recent evidence has also established the existence of an ATP-dependent phase of RecA-mediated DNA strand exchange. ATP hydrolysis renders the reaction unidirectional and permits the bypass of structural barriers in the DNA during recombinational DNA repair. Two competing models for how ATP hydrolysis might be coupled to DNA strand exchange will be tested by more accurately defining the kinetic parameters of DNA strand exchange, and by examining reactions with new DNA substrates that address key experimental predictions. Selected mutant RecA proteins with diminished ATPase function will be characterized.
| 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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| Chen, Stefanie H; Byrne-Nash, Rose T; Cox, Michael M (2016) Escherichia coli RadD Protein Functionally Interacts with the Single-stranded DNA-binding Protein. J Biol Chem 291:20779-86 |
| 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 |
| Gruber, Angela J; Olsen, Tayla M; Dvorak, Rachel H et al. (2015) Function of the N-terminal segment of the RecA-dependent nuclease Ref. Nucleic Acids Res 43:1795-803 |
| Robinson, Andrew; McDonald, John P; Caldas, Victor E A et al. (2015) Regulation of Mutagenic DNA Polymerase V Activation in Space and Time. PLoS Genet 11:e1005482 |
| 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 |
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