The goal of this research project is to determine how the recA protein of E. coli utilizes the energy of ATP hydrolysis to promote DNA strand pairing reactions during homologous genetic recombination and recombinational DNA repair. Using transient kinetic analysis, the elementary steps on the recA protein-catalyzed ssDNA-dependent ATP hydrolysis reaction pathway have been determined and the rate- determining step on the reaction pathway has been identified as an ATP- mediated isomerization of the recA-ssDNA complex. Moreover, by examining alternate nucleotide cofactors, it has been established that nucleoside triphosphate hydrolysis is not, in itself, sufficient to drive the strand exchange reaction; a nucleoside triphosphate must also be able to allosterically stabilize the recA-ssDNA complex in a strand exchange-active conformational state. The research described in this competing renewal application will continue our investigations of the biochemical properties of the recA-ssDNA complex and the role of ATP- mediated conformational changes on the DNA strand exchange reaction pathway. The specific goals for the next funding period are: 1. To refine and extend our kinetic mechanism for the ssDNA-dependent ATP hydrolysis reaction. 2. To identify the active site interactions between the recA protein and ATP that control the conformational state of the recA-ssDNA complex. 3. To determine the structural origin and mechanistic function of the rate-determining isomerization of the recA-ssDNA complex. 4. To elucidate the mechanistic nature of the coupling between ATP hydrolysis and DNA branch migration.
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