Double strand DNA break repair in D. radiodurans

描述(申请人提供)：这项研究计划探索耐辐射球菌(DR)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抗辐射能力最强的微生物中的一个小家族的一部分。在5000灰剂量的g辐射导致数百条双链断裂后，这种细菌的染色体在几个小时内重新组装，这是DNA新陈代谢的一项非凡壮举，没有造成致命或诱发突变。这一强大的DSB修复过程将在体内和体外进行探索，主要目标是详细了解修复途径及其所涉及的酶。这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，纯化参与修复的蛋白质，表征这些蛋白质，阐明途径中的蛋白质-蛋白质相互作用，以及继续发展一个明确的体外系统来促进DSB修复。这项工作将利用耐辐射球菌的完整基因组序列，以及最近对耐辐射球菌基因组微阵列的工作，该微阵列已经确定了由高水平梯度诱导的基因。这项工作将由四个拥有互补技能和经验的实验室组成的联盟合作进行：迈克尔·考克斯、约翰·巴蒂斯塔、詹姆斯·凯克和罗斯·英曼。巴蒂斯塔实验室已经进行了微阵列分析，并开发了方便地创建葡萄球菌基因敲除的工具。考克斯和凯克实验室带来了DNA修复过程的酶学经验和背景。英曼实验室用电子显微镜研究DNA新陈代谢。对几种奇球菌蛋白的初步研究，如DR RecA蛋白，已经产生了令人惊讶的结果，这表明了对奇球菌DNA修复过程进行研究的可能性。一些关键的重组酶，如recB、recC和Ress，在DR基因组中缺失，这表明DSB修复的主要途径在葡萄球菌中是不同于在其他细菌中的。这项工作有可能识别出DNA修复的全新蛋白质和途径。

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.