Abstract

The goal of the 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 strand pairing reactions that are carried out by DNA recombinases during homologous genetic recombination. In particular, the mechanism of the protypical DNA recombinase, the recA protein of Escherichia coli, will be analyzed. Detailed mechanistic models will be developed for the various elementary reactions of the recA protein that together constitute a recA protein-promoted strand invasion event. In addition, site-directed mutagenesis will be used, in combination with sequence analysis and chemical modification, to create new mutant recA proteins that will be used to test these mechanistic models. These mutant proteins will also be analyzed in the more complex strand exchange reactions in order to determine how specific well-defined defects in the various elementary reactions of the recA protein affect the overall strand exchange process. These studies will lead to an understanding of the mechanistic principles that are involved in an ATP-dependent DNA recombinase- promoted strand pairing event.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036516-08
Application #
3290630
Study Section
Biochemistry Study Section (BIO)
Project Start
1986-05-01
Project End
1994-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
8
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
Johns Hopkins University
Department
Type
Schools of Public Health
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218

  Publications

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

Showing the most recent 10 out of 28 publications