Generation and Repair of an Unusual UV Photoproduct

不寻常的 UV Photoproduct 的生成和修复

• 批准号: 7056688
• 负责人: Joan B Broderick
• 金额: $ 24.87万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7056688
• 关键词: DNA damage; DNA footprinting; DNA repair; S adenosylmethionine; X ray; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme mechanism; gel mobility shift assay; intermolecular interaction; lyase; protein binding; protein structure function; ultraviolet radiation

DESCRIPTION (provided by applicant): Repair of UV-induced DNA damage is central to the prevention of a number of adverse conditions such as melanoma, but in most cases the mechanism of such DNA repair is not well understood at a molecular level. Under normal cellular conditions, the major DNA photoproducts of UV irradiation are cyclobutane pyrimidine dimers (TT, CT, and CC dimers) and 6,4-photoproducts. 5-Thyminyl-5,6-dihydrothymine is typically a minor product of UV irradiation. This typically minor UV photoproduct becomes the major UV photoproduct under certain conditions, however, including the conditions that exist in bacterial spores (thus the common name is spore photoproduct, SP). Remarkably, the formation of spore photoproduct is correlated with the unusually high resistance of bacterial spores to UV irradiation, and this resistance arises in part from the novel DNA repair enzyme that repairs SP. The overall goals of this proposal include investigating the mechanism by which the repair enzyme, SP lyase, recognizes and repairs SP. In addition, experiments designed to probe the mechanism by which SP is formed at the expense of cyclobutane pyrimidine dimers, are described. The specific aims of this proposal are as follows: 1) To investigate the structure and environment of the iron-sulfur cluster in spore photoproduct lyase using appropriate spectroscopic methods; 2) To probe the mechanistic details of the DNA repair reaction using isotope labeling and substrate analogs; 3) To investigate the molecular basis of the interaction of SPL with UV-damaged DNA using gel-shift assays and DNA footprinting; 4) To investigate the interaction between the iron-sulfur cluster of SP lyase and its substrates S-adenosylmethionine and spore photoproduct using Mossbauer, electron-nuclear double resonance, and electron-spin echo envelope modulation; and 5) To investigate the structural basis of SP formation using biochemical and X-ray structural methods.

描述（由申请人提供）：修复紫外线引起的 DNA 损伤对于预防许多不良状况（如黑色素瘤）至关重要，但在大多数情况下，这种 DNA 修复的机制在分子水平上尚不清楚。在正常细胞条件下，紫外线照射的主要DNA光产物是环丁烷嘧啶二聚体（TT、CT和CC二聚体）和6,4-光产物。 5-胸腺嘧啶基-5,6-二氢胸腺嘧啶通常是紫外线照射的次要产物。然而，在某些条件下，包括细菌孢子中存在的条件（因此通用名称为孢子光产物，SP），这种通常次要的紫外光产物会成为主要的紫外光产物。值得注意的是，孢子光产物的形成与细菌孢子对紫外线照射的异常高的抵抗力有关，这种抵抗力部分来自于修复 SP 的新型 DNA 修复酶。该提案的总体目标包括研究修复酶 SP 裂合酶识别和修复 SP 的机制。此外，还描述了旨在探索以环丁烷嘧啶二聚体为代价形成 SP 的机制的实验。该提案的具体目标如下： 1）利用适当的光谱方法研究孢子光产物裂合酶中铁硫簇的结构和环境； 2）利用同位素标记和底物类似物探索DNA修复反应的机制细节； 3) 使用凝胶迁移分析和 DNA 足迹法研究 SPL 与 UV 损伤 DNA 相互作用的分子基础； 4) 利用穆斯堡尔、电子核双共振和电子自旋回波包络调制研究SP裂解酶的铁硫簇与其底物S-腺苷甲硫氨酸和孢子光产物之间的相互作用； 5) 利用生化和X射线结构方法研究SP形成的结构基础。