This research proposal explores the DNA repair pathways of the bacterium Deinococcus radiodurans, (Dr) focusing on double strand break (DSB) repair. D. radiodurans is part of a small family of bacterial species that are among the most radiation-resistant organisms known. After a 5000 gray dose of g radiation generating hundreds of double strand breaks, this bacterium's chromosomes are reassembled over a few hours in a remarkable feat of DNA metabolism, resulting in no lethality or induced mutation. This robust DSB repair process will be explored both in vivo and in vitro, with a detailed molecular understanding of the repair pathways and the enzymes involved in them being the major goal. The five specific aims encompass a systematic effort to identify important enzymatic functions, define repair pathways, purify proteins involved in repair, characterize those proteins, elucidate protein-protein interactions within the pathways, and continue the development of a defined in vitro system to promote DSB repair. The work will take advantage of the completed genomic sequence of Deinococcus radiodurans, as well as recent work with Deinococcus genomic microarrays that has identified genes induced by high levels of gradiation. The work will be carried out cooperatively by a consortium of four laboratories with complementary skills and experience: Michael Cox, John Battista, James Keck, and Ross Inman. The Battista laboratory has carried out the microarray analysis, and has developed tools for the convenient creation of gene knockouts in Deinococcus. The Cox and Keck laboratories bring experience and background in the enzymology of DNA repair processes. The Inman laboratory explores DNA metabolism with electron microscopy. Nascent work on a few Deinococcus proteins, such as the Dr RecA protein, have already generated surprises that speak to the potential of an investigation of Deinococcus DNA repair processes. Some key recombination enzymes, such as recB, recC, and recE, are absent in the Dr genome, indicating that the predominant pathways for DSB repair in Deinococcus are distinct from those that dominate in other bacteria. The work has the potential for the identification of entirely novel proteins and pathways for DNA repair.
