Double strand DNA break repair in D. radiodurans

D. radiodurans 中的双链 DNA 断裂修复

• 批准号: 7171806
• 负责人: Michael M. Cox
• 金额: $ 31.48万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7171806
• 关键词: Bacteria; Basic Science; Biochemical; Bioremediations; Biotechnology; Cells; Chromosomes; Classification; Coupled; Crude Extracts; Cytolysis; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; Deinococcus; Deinococcus radiodurans; Development; Dose; Double Strand Break Repair; Electron Microscopy; Enzymes; Family; Genes; Genetic; Genetic Recombination; Genome; Genomics; Goals; Gray unit of radiation dose; Hand; Hour; In Vitro; Indium; Individual; Induced Mutation; Investigation; Ionizing radiation; Laboratories; Lead; Literature; Measurable; Mediating; Metabolism; Methods; Microarray Analysis; Molecular; Mutation; Organism; Pathway interactions; Phase; Play; Process; Property; Proteins; Pulsed-Field Gel Electrophoresis; Radiation; Radioactive Waste; Range; Rec A Recombinases; RecQ protein; Repair Enzymology; Reporting; Research; Research Personnel; Research Proposals; Resistance; Role; Source; Staging; System; Testing; Work; design; enzyme pathway; experience; helicase; in vitro Assay; in vivo; insight; interest; irradiation; knockout gene; novel; nuclease; practical application; prevent; protein protein interaction; protein purification; radiation resistance; reconstitution; repaired; research study; restoration; skills; tool

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.

描述（由申请人提供）：本研究建议探讨了迪那核细菌的DNA修复途径，（DR）重点是双链断裂（DSB）修复。 D. radiodurans是一小部分细菌物种的一部分，它们是最耐辐射的生物之一。在5000个灰剂量的G辐射产生了数百链断裂后，该细菌的染色体在几个小时内重新组装了几个小时，以显着的DNA代谢，导致无致命性或诱导的突变。这种强大的DSB修复过程将在体内和体外探索，并具有对修复途径的详细分子理解，而涉及的酶是主要目标。五个特定的目的包括一个系统的努力，以识别重要的酶促功能，定义修复途径，纯化蛋白质涉及维修，表征这些蛋白质，阐明途径中的蛋白质 - 蛋白质相互作用，并继续开发定义的体外系统以促进DSB维修。这项工作将利用放射性甲虫的完整基因组序列，以及最近与Deinococcus Genococcus基因组微阵列的工作，这些阵列已经确定了由高渐变诱导的基因。这项工作将由四个具有互补技能和经验的实验室的财团合作进行：迈克尔·考克斯，约翰·巴蒂斯塔，詹姆斯·凯克和罗斯·伊曼。 Battista实验室已经进行了微阵列分析，并开发了用于方便地创建Deinococcus基因敲除的工具。考克斯和凯克实验室为DNA修复过程的酶学带来了经验和背景。 Inman实验室探索了通过电子显微镜的DNA代谢。在一些Deinococcus蛋白（例如RECA蛋白）上的新生作品已经产生了惊喜，这说明了对DEINOCOCCUS DNA修复过程的研究的潜力。 DR基因组中没有一些关键的重组酶，例如RECB，RECC和RECE，表明DEINOCOCCUS中DSB修复的主要途径与其他细菌占主导地位的途径不同。这项工作具有鉴定完全新颖的蛋白质和DNA修复途径的潜力。