Double strand DNA break repair in D. radiodurans

描述（由申请人提供）：本研究计划探索耐辐射球菌（Dr）细菌的DNA修复途径，重点是双链断裂（DSB）修复。耐辐射细菌是已知的最耐辐射的生物之一。在5000灰色剂量的g辐射产生数百条双链断裂后，这种细菌的染色体在DNA代谢的非凡壮举中在几个小时内重新组装，不会导致致命或诱导突变。这种强大的DSB修复过程将在体内和体外进行探索，对修复途径和其中涉及的酶进行详细的分子理解是主要目标。五个具体目标包括系统地识别重要的酶功能，定义修复途径，纯化修复中涉及的蛋白质，表征这些蛋白质，阐明途径内蛋白质-蛋白质相互作用，并继续发展一个明确的体外系统来促进DSB修复。这项工作将利用已完成的耐辐射球菌基因组序列，以及最近对耐辐射球菌基因组微阵列的研究，该微阵列已经确定了由高水平梯度诱导的基因。这项工作将由四个具有互补技能和经验的实验室组成的联盟合作进行：Michael Cox、John Battista、James Keck和Ross Inman。巴蒂斯塔实验室进行了微阵列分析，并开发了方便地在球菌中创建基因敲除的工具。考克斯和凯克实验室带来了DNA修复过程酶学方面的经验和背景。英曼实验室用电子显微镜研究DNA代谢。对一些Deinococcus蛋白的初步研究，如Dr RecA蛋白，已经产生了令人惊讶的结果，说明了对Deinococcus DNA修复过程的研究潜力。一些关键的重组酶，如recB， recC和recE，在Dr基因组中不存在，这表明在Deinococcus中DSB修复的主要途径与在其他细菌中占主导地位的途径不同。这项工作有可能鉴定出全新的蛋白质和DNA修复途径。

DESCRIPTION (provided by applicant): 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.