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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. Perhaps most important, homologous genetic recombination plays a key role in DNA damage tolerance and in the resolution of stalled and collapsed replication forks. The goal of this laboratory is to understand how these genetic rearrangements come about, and how they interface with other aspects of DNA metabolism. The work is carried out using bacteria as model organisms. The approaches used in the lab vary from classical Genetics to Bioinformatics to basic Enzymology to Structural Biology. Technologies unavailable in the laboratory are accessed via an extensive network of collaborations. Currently, there are three areas of active investigation: (1) RecA protein filaments on DNA as barriers to DNA replication; (2) the function and mechanism of several enzymes involved in DNA double strand break repair, including RarA/MgsA, Uup, RadA, RadD, RecG, RecF, RecO, and RecR; and (3) the molecular basis of extreme resistance to ionizing radiation.

All processes in DNA metabolism: replication, repair, recombination, and transcription, all share the same DNA substrate. Evolution must balance these processes to minimize conflicts. 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 functional form of the protein is a RecA filament that forms on the DNA. In principle, these filaments represent the largest barrier that a replication fork may encounter on its DNA substrate. RecA filaments must thus exist only transiently on DNA. We have generated RecA proteins with an enhanced capacity to promote recombinational processes. However, these enhanced function RecA variants bind to DNA more persistently and slow cell growth due to conflicts with DNA replication. We are exploring the mechanisms employed by the cell to deal with these conflicts.

The RarA/MgsA, Uup, and RadD proteins are all demonstrably important for cell survival after treatments that result in chromosomal strand breaks. However, their functions have been enigmatic. Our approach to elucidating the function and mechanism of action of these proteins is multidisciplinary, with key contributions arising by collaboration. All of these proteins are likely to play important roles at the interfaces of different processes in DNA metabolism.

Our interest in extreme resistance to ionizing radiation (IR) evolved from 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 the effects of IR than is E. coli. The tolerance reflects, at least in part, a robust repair of double strand breaks. Our early effort to study Deinococcus proteins has gradually been replaced by a broader effort to understand the mechanisms underlying extreme IR resistance. Our approach in this case is to utilize directed evolution. We have taken wild type E. coli cells that are not IR resistant and are subjecting them to iterative rounds of irradiation (enough to kill 99+% of the population) followed by outgrowth of survivors. The result is the generation of E. coli populations with ever increasing levels of IR resistance. Deep sequencing of these populations allows us to follow the appearance of mutations that contribute to the phenotype. This is a long-term experiment, with the goal of generating E. coli populations with IR resistance exceeding what is observed in Deinococcus. Identifying the mutations and mechanisms for IR resistance that arise in these populations represents a broad effort involving Bioinformatics, Genetics, and Biochemistry.

A broad approach to each of these problems is facilitated by active collaborations with 6-10 research groups around the world at any given time.