CURRENT RESEARCH

Genetic recombination is perhaps the least understood of all the processes that contribute to DNA metabolism. Many classes of DNA rearrangements occur in all cells and play important roles in gene regulation, development carcinogenesis, and evolution. The goal of this laboratory is to understand how these genetic rearrangements come about. The approach is to study in detail the isolated enzymes that play central roles in different classes of genetic recombination events. Currently, we are working on the enzymology and molecular biology of the RecA protein of bacteria, the reconstitution of complex recombinational repair processes in vitro with purified enzymes, and the enzymology of eukaryotic recombination enzymes. A major theme of all of these studies is the increasingly evident links between recombination and other aspects of DNA metabolism, particularly replication.

The RecA protein is the key component required for recombinational DNA repair in bacteria. This protein is capable of pairing two homologous molecules of DNA, exchanging strands of DNA between them. The reaction occurs in several phases that are easily distinguished experimentally. Our efforts are directed at a determination of the mechanism by which complexes of RecA protein bound to DNA promote a unidirectional DNA strand exchange reaction coupled to ATP hydrolysis, and biophysical studies aimed at understanding the formation, disassembly and structure of RecA filaments on DNA. The eukaryotic homologue of the RecA protein is Rad51, and we are now studying this protein as well as several other eukaryotic proteins (Rad52, Rad54, etc) that interact with it as a complement to our RecA work. We are also studying several other E. coli proteins involved in recombinational DNA repair and the repair of stalled replication forks. These include the RecF, RecO, and RecR proteins, which function early in recombinational processes, and the RuvA and RuvB proteins which function late. The RecF, O, and R proteins appear to act together to regulate the formation and disassembly of RecA protein complexes on DNA. The RuvA and B proteins form a complex that plays a key role in processing branched DNA recombinational intermediates. All of these proteins currently present a variety of challenging mechanistic questions.

We have recently initiated a new project in the lab to explore the mechanism of double strand break repair in the bacterium Deinococcus radiodurans. This bacterium is part of a small family that includes the most radiation resistant organisms known. Deinococcus can withstand 1.7 Mrads of cobalt-60 radiation (enough to turn Pyrex glassware to dust) without lethality or induced mutation. This extraordinary radiation resistance reflects an unusually robust act of DNA repair systems. We have several components of the double strand break repair system under study, and are working to reconstitute the entire system with purified enzymes in vitro.