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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM036516-16S1
Application #
6585684
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Ikeda, Richard A
Project Start
1986-05-01
Project End
2002-11-30
Budget Start
2001-05-01
Budget End
2002-11-30
Support Year
16
Fiscal Year
2002
Total Cost
$195,824
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Public Health
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Nayak, Sunil; Bryant, Floyd R (2015) Kinetics of the ATP and dATP-mediated formation of a functionally-active RecA-ssDNA complex. Biochem Biophys Res Commun 463:1257-61
Grove, Diane E; Anne, Geetha; Hedayati, Mohammad A et al. (2012) Stimulation of the Streptococcus pneumoniae RecA protein-promoted three-strand exchange reaction by the competence-specific SsbB protein. Biochem Biophys Res Commun 424:40-4
Steffen, Scott E; Bryant, Floyd R (2012) Altered nucleotide cofactor-dependent properties of the mutant [S240K]RecA protein. Biochem Biophys Res Commun 421:527-31
Katz, Francine S; Bryant, Floyd R (2003) Three-strand exchange by the Escherichia coli RecA protein using ITP as a nucleotide cofactor: mechanistic parallels with the ATP-dependent reaction of the RecA protein from Streptococcus pneumoniae. J Biol Chem 278:35889-96
Steffen, Scott E; Katz, Francine S; Bryant, Floyd R (2002) Complete inhibition of Streptococcus pneumoniae RecA protein-catalyzed ATP hydrolysis by single-stranded DNA-binding protein (SSB protein): implications for the mechanism of SSB protein-stimulated DNA strand exchange. J Biol Chem 277:14493-500
Hedayati, Mohammad A; Steffen, Scott E; Bryant, Floyd R (2002) Effect of the Streptococcus pneumoniae MmsA protein on the RecA protein-promoted three-strand exchange reaction. Implications for the mechanism of transformational recombination. J Biol Chem 277:24863-9
Katz, F S; Bryant, F R (2001) Interdependence of the kinetics of NTP hydrolysis and the stability of the RecA-ssDNA complex. Biochemistry 40:11082-9
Steffen, S E; Bryant, F R (2001) Purification and characterization of the single-stranded DNA binding protein from Streptococcus pneumoniae. Arch Biochem Biophys 388:165-70
Nayak, S; Hildebrand, E L; Bryant, F R (2001) ADP-dependent DNA strand exchange by the mutant [P67G/E68A] RecA protein. J Biol Chem 276:14933-8
Steffen, S E; Bryant, F R (2000) Purification and characterization of the RecA protein from Streptococcus pneumoniae. Arch Biochem Biophys 382:303-9

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