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.
