Homologous genetic recombination is a fundamental biological process that is well appreciated at a formal genetic level: it plays central roles in inheritance and the proper segregation of chromosomes, but is also important in DNA repair, replication, regulation, and differentiation. In addition, the specific introduction of foreign DNA into eukaryotic chromosomes a basic step in the developing are of gene therapy, is critically dependent on genetic recombination machinery. The RecA protein of E. coli has served as a recombinase prototype, and has been the most widely investigated member of this universal class of proteins. Although RecA has been the subject of biological research for 30 years, the molecular mechanisms of the genetic exchange events remain unclear. The long-term goal of the research proposed herein is to understand in molecular terms the structural and dynamic mechanisms of genetic recombination, the processes by which the genetic information is reorganized by the physical rearrangement of elemental pieces of the genetic material. During the project period to be supported by an NIH Research Grant, the research is directed towards significant and novel contributions to the molecular understanding of RecA-mediated recombination by characterizing the forces of molecular recognition that control recombination events. Bio-organic and biophysical approaches will be used to examine the equilibrium and kinetics of the interactions that are important for recombination. In order to dissect the molecular steps involved in recombination, our initial efforts will focus on key early events: DNA binding and the search for homology between substrate DNAs. Because recombination is an active process involving elusive intermediary protein-DNA complexes, the research will focus a major emphasis on a presteady-state kinetic approach using stopped-flow agents and site- directed mutagenesis will elucidate the molecular mechanisms by which RecA binds DNA an allows sequence homology to be recognized. Ultimately, we hope to exploit this understanding in vivo through the control and directed manipulation of RecAs function.
