Double strand DNA break repair in D. radiodurans

描述（由申请人提供）：本研究计划探讨了耐辐射奇球菌（Deinococcus radiodurans）的DNA修复途径，（博士）专注于双链断裂（DSB）修复。D.耐辐射菌是已知最耐辐射的生物体之一的细菌物种小家族的一部分。在产生数百个双链断裂的5000戈瑞剂量的辐射后，这种细菌的染色体在几个小时内以DNA代谢的非凡壮举重新组装，导致没有致死性或诱导突变。这种强大的DSB修复过程将在体内和体外进行探索，主要目标是对修复途径和参与其中的酶进行详细的分子理解。这五个具体目标包括系统性的努力，以确定重要的酶功能，确定修复途径，纯化参与修复的蛋白质，表征这些蛋白质，阐明蛋白质-蛋白质相互作用的途径，并继续发展一个明确的体外系统，以促进DSB修复。这项工作将利用已完成的耐辐射异常球菌基因组序列，以及最近的工作与异常球菌基因组微阵列，已确定基因诱导的高水平的梯度。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：迈克尔考克斯，约翰巴蒂斯塔，詹姆斯凯克和罗斯英曼。Battista实验室已经进行了微阵列分析，并开发了用于在异常球菌中方便地创建基因敲除的工具。考克斯和凯克实验室带来了DNA修复过程酶学的经验和背景。英曼实验室用电子显微镜探索DNA代谢。对一些异常球菌蛋白质（如Dr RecA蛋白质）的研究已经产生了令人惊讶的结果，这说明了对异常球菌DNA修复过程进行研究的潜力。一些关键的重组酶，如recB、recC和recE，在Dr基因组中不存在，这表明异常球菌中DSB修复的主要途径与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.