Generation and Repair of an Unusual UV Photoproduct

描述（由申请人提供）：UV诱导的DNA损伤的修复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还没有得到很好的理解。在正常细胞条件下，UV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UV照射的次要产物。这种典型的次要UV光产物在某些条件下变成主要UV光产物，然而，包括存在于细菌孢子中的条件（因此，通用名称是孢子光产物，SP）。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抗性相关，并且这种抗性部分来自修复SP的新型DNA修复酶。该提议的总体目标包括研究修复酶SP裂解酶识别和修复SP的机制。此外，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。本论文的主要目的是：1）利用光谱学方法研究孢子光产物裂解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研究DNA修复反应的机理，3）利用凝胶迁移分析和DNA足迹法研究SPL与紫外损伤DNA相互作用的分子基础，4）利用分子生物学方法研究SPL与紫外损伤DNA相互作用的分子机制。4）利用穆斯堡尔谱、电子-核双共振和电子-自旋回波包络调制技术研究SP裂解酶的铁硫簇与底物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。

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.