The goal of this proposed research is to determine the molecular mechanisms by which enzymes utilize the free energy of ATP hydrolysis to bring about changes in the structure of the DNA helix. The research will focus on the DNA pairing and unpairing reactions that occur during homologous genetic recombination. The mechanisms of action of two proteins from E. coli will be analyzed: the recA protein and the uvrD protein. The recA protein is responsible for the pairing of DNA molecules in new hybrid combinations during homologous recombination. In this research, several new DNA pairing reactions that are designed to elucidate the individual steps that comprise a strand invasion event will be investigated. The general mechanism of the recA protein-promoted homologous alignment of DNA chains will be determined and the role of ATP hydrolysis in the process will be defined. In a second project, the molecular mechanism of the ATP-dependent, DNA helicase-catalyzed unwinding of duplex DNA will be studied. The research will initially focus on the uvrD protein, which has been implicated in DNA recombination and repair. The methodology that is developed will be applicable to the mechanistic study of DNA helicases in general. A careful study of the mechanisms of action of individual enzymes will provide a basis for studying the coordinate action of multiprotein systems during the course of a complete recombination event.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036516-03
Application #
3290626
Study Section
Biochemistry Study Section (BIO)
Project Start
1986-05-01
Project End
1989-04-30
Budget Start
1988-05-01
Budget End
1989-04-30
Support Year
3
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
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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