Molecular biology and enzymology of genetic recombination and DNA repair
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, two systems are under investigation: recombinational DNA repair in E. coli and the extraordinary repair of double strand breaks during chromosome restoration in the bacterium Deinococcus radiodurans after heavy doses of ionizing radiation.
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: 1) a study of the structure of a putative 3-stranded DNA pairing intermediate, and 2) 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. The system offers a variety of unique problems on protein-nucleic acid interactions, unusual DNA structures, and biochemical energetics.
In addition to RecA, we are studying several other E. coli proteins involved in recombinational DNA repair. Our present focus is on proteins that modulate RecA function. These include the RecF, RecO, and RecR proteins, which function early in recombinational processes, and the DinI and RecX proteins, which seem to play a role in modulating RecA function during the SOS response. The RecF, O, and R proteins appear to act together to regulate the formation and disassembly of RecA protein complexes on DNA. The DinI and RecX proteins modulate also the assembly and disassembly of RecA filaments, and may affect the function of assembled filaments. All of these proteins currently present a variety of challenging mechanistic questions.
Our newest enterprise is an effort to examine the facile repair of chromosomes in the radiation-resistant bacterium Deinococcus radiodurans. This organism is several hundred times more resistant to radiation than is E. coli. The tolerance reflects, at least in part, a robust repair of double strand breaks. The mechanisms by which this is effected are being actively investigated, focusing on proteins (many of them novel) with an established role in the repair processes.
A broad approach to each of these problems is facilitated by active collaborations with over a half dozen research groups around the world.