This research will be done primarily in St. Petersburg, Russia at Petersburg Nuclear Physics Institute in collaboration with Vladislav Lanzov as an extension of NIH grant Number R01GM32335. 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 R01 GM32335 (Cox) is a detailed understanding of RecA-mediated DNA strand exchange. The hypothesis that recombinational DNA repair is the primary function of RecA protein in vivo provides an intellectual framework. One of the specific aims of GM32335-21 (recently funded) is to understand how RecA function is autoregulated. Pseudomonas aeruginosa, an important human pathogen, is the source of a RecA protein that we will investigate to further this aim. We propose to continue a productive collaboration that has been extending our understanding of RecA autoregulation through an analysis of the Pseudomonas aeruginosa RecA protein. Work to date has provided numerous mechanistic and structural insights that we will build on. Together with the laboratory of Dr. Vladislava Lanzov in St. Petersburg, Russia, we will further explore the biochemistry of the P. aeruginosa RecA protein, and define the enzymatic differences between it and the E. coli RecA. We have pinpointed likely regions of the protein responsible for autoregulation and for certain other functional differences between the RecA proteins. The new work should help test key features of models for RecA-mediated DNA strand exchange, and may also help identify RecA variants with enhanced DNA binding and strand exchange functions. Such proteins may eventually prove useful in efforts to use RecA in gene therapy protocols and to generate crystals of RecA-DNA complexes for structural analysis.

Agency
National Institute of Health (NIH)
Institute
Fogarty International Center (FIC)
Type
Small Research Grants (R03)
Project #
2R03TW001319-04
Application #
6831418
Study Section
International and Cooperative Projects 1 Study Section (ICP)
Program Officer
Sina, Barbara J
Project Start
2000-01-01
Project End
2007-07-31
Budget Start
2004-08-01
Budget End
2005-07-31
Support Year
4
Fiscal Year
2004
Total Cost
$39,020
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Bakhlanova, Irina V; Dudkina, Alexandra V; Baitin, Dima M et al. (2010) Modulating cellular recombination potential through alterations in RecA structure and regulation. Mol Microbiol 78:1523-38
Baitin, Dmitry M; Bakhlanova, Irina V; Chervyakova, Darya V et al. (2008) Two RecA protein types that mediate different modes of hyperrecombination. J Bacteriol 190:3036-45
Rychkov, Georgy; Petukhov, Michael (2007) Joint neighbors approximation of macromolecular solvent accessible surface area. J Comput Chem 28:1974-89
Kaboev, Oleg; Luchkina, Ludmila; Shalguev, Valery et al. (2006) Improved RecA-assisted fluorescence assay for DNA strand exchange reaction. Biotechniques 40:736, 738
Petukhov, Michael; Lebedev, Dmitry; Shalguev, Valery et al. (2006) Conformational flexibility of RecA protein filament: transitions between compressed and stretched states. Proteins 65:296-304
Lanzov, Vladislav A; Bakhlanova, Irina V; Clark, Alvin J (2003) Conjugational hyperrecombination achieved by derepressing the LexA regulon, altering the properties of RecA protein and inactivating mismatch repair in Escherichia coli K-12. Genetics 163:1243-54
Lebedev, D V; Baitin, D M; Islamov, A Kh et al. (2003) Analytical model for determination of parameters of helical structures in solution by small angle scattering: comparison of RecA structures by SANS. FEBS Lett 537:182-6
Baitin, Dmitry M; Zaitsev, Eugene N; Lanzov, Vladislav A (2003) Hyper-recombinogenic RecA protein from Pseudomonas aeruginosa with enhanced activity of its primary DNA binding site. J Mol Biol 328:1-7
Bakhlanova, I V; Ogawa, T; Lanzov, V A (2001) Recombinogenic activity of chimeric recA genes (Pseudomonas aeruginosa/Escherichia coli): a search for RecA protein regions responsible for this activity. Genetics 159:7-15
Chervyakova, D; Kagansky, A; Petukhov, M et al. (2001) [L29M] substitution in the interface of subunit-subunit interactions enhances Escherichia coli RecA protein properties important for its recombinogenic activity. J Mol Biol 314:923-35