In living organisms, there is a dynamic balance between the need to preserve genomic information and the need to generate genetic diversity. The repair of damaged DNA is essential to the maintenance of heritable genetic information, while the variation of that information drives evolutionary adaptation. The E. coli RecA protein mediates events in both pathways, and because its functions are conserved from bacteriophage to humans, its study has provided a paradigm for understanding these essential biological processes. The molecular mechanisms underlying the complex process of DNA strand pairing and exchange are not yet understood. In particular, there is still substantial uncertainty with regard to the role of the sequence-dependent DNA-DNA interactions that are the hallmark of homologous recombination. In order to delimit possible models for recombinational activities, we propose to investigate the mechanism of the DNA strand exchange reaction mediated by the RecA protein. During the project period to be supported by an NIH Research Grant, the research is directed towards the characterization of site-specific interactions among the reaction constituents in order to reach a molecular understanding of recombination and recombinational repair by the construction and elaboration of three-dimensional models of the intermediates in the reactions. Bioorganic and biophysical approaches will be used to examine the equilibrium and kinetics of the interactions that are important for recombination. In order to tease apart the molecular events, 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 spectrofluoriometry. In combination with affinity labeling, time-resolved FRET structural characterization, and EPR dynamic characterization, transient kinetics agents will elucidate the molecular mechanisms by which RecA binds DNA and allows sequence homology to be recognized. Ultimately, we hope to exploit this understanding in vivo through the control and directed manipulation of RecA's function.
