The laboratory is examining the RecA, RecBCD, and SSB proteins, of E. coli and the Rad51, Rad52, and RP-A proteins of S. cerevisiae. The goals of our work are both to establish in vitro systems that accurately reproduce the cellular process and to understand the biochemical function of each participant in these complex reactions.
The RecA protein promotes a reaction that is both biochemically unique and central to the recombination process, namely, the ATP-dependent homologous pairing and exchange of DNA strands; despite considerable study, many biochemical aspects of this reaction remain unclear, including the mechanisms of both homologous DNA recognition and energy transduction.
The RecBCD enzyme is a DNA helicase that is also an ATP-dependent nuclease. It interacts with a specific DNA sequence, essential for elevated recombination activity, referred to as Chi site. Through an unknown biochemical mechanism, Chi sites attenuate the nuclease activity, but not the helicase activity, of RecBCD enzyme.
The SSB protein is a single-stranded DNA binding protein that stimulates the activities of both the RecA and RecBCD proteins by virtue of its ability to bind ssDNA. Proteins with similar activities have been found in eukaryotes as well.
The Rad51 protein shows considerable sequence similarity to the RecA protein. Similarly, the RP-A protein is presumably the functional equivalent of the SSB protein. The manner by which the activities of these proteins are coordinated to produce novel product DNA molecules is being examined.
The experimental approaches used include the thermodynamic, kinetic, and structural techniques. We have used fluorescence, UV, and infrared spectroscopy, atomic force microscopy (AFM), stopped-flow kinetics, spectroscopic and electrophoretic assays for enzymatic activity, protein-nucleic acid crosslinking, chemical modification, electron microscopy, video-enhanced fluorescence microscopy, optical trapping, site-specific mutagenesis, and in vivo assays for protein-protein interactions.