Molecular mechanism of genetic recombination




  Our research is focused on the molecular mechanisms of genetic recombination, with the long-term objective being the reconstitution of in vitro systems that accurately reproduce the cellular processes. We are characterizing the biochemical properties of proteins essential to homologous recombination, in prokaryotes, eukaryotes, and Archaea. DNA Repair
   In E. coli, the RecA, RecBCD, RecQ, RuvABC, and SSB proteins, and a specific DNA sequence called Chi, are essential to homologous recombination. The RecA protein possesses the unique ability to pair homologous DNA molecules and to promote the subsequent exchange of DNA strands. Since RecA protein is the prototypic DNA strand exchange protein, we are interested in the biochemical mechanism of protein-mediated recognition and exchange of homologous DNA strands. The RecBCD enzyme is both a DNA helicase and a nuclease with the remarkable properties that its nuclease activity, but not its helicase activity, is attenuated by interaction with the Chi sequence, and that it will actively load RecA protein onto ssDNA. RecQ protein is a helicase that can also effect recombination events. SSB protein is an ssDNA binding protein that stimulates the activities of RecA, RecBCD, and RecQ proteins by virtue of its ability to bind ssDNA. Recently, we reconstituted an in vitro pairing reaction that requires the concerted action of each of these proteins; the role of each protein in this reaction is under investigation. RecA protein-dsDNA complex imaged by atomic force microscopy (AFM):COVER.TIF (266940 bytes)
  We are also studying the biochemistry of homologous recombination in the yeast, S. cerevisiae and the archaeon, S. solfataricus. Rad51 and RadA proteins are the RecA protein homologues, respectively. In yeast, at least three ancillary proteins are needed for Rad51 protein-mediated DNA strand exchange: these include the RP-A, Rad52, and Rad54 proteins. We are studying the mechanism of these reconstituted reactions.