Generation and Repair of an Unusual UV Photoproduct

描述(申请人提供)：修复紫外线诱导的DNA损伤是预防一些不利条件如黑色素瘤的核心，但在大多数情况下，这种DNA修复的机制在分子水平上还不是很清楚。在正常细胞条件下，紫外光照射DNA的主要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-5，6-二氢胸腺嘧啶通常是紫外线辐射的次要产物。然而，在某些条件下，这种通常很小的紫外光产物会成为主要的紫外光产物，包括细菌孢子中存在的条件(因此，通常的名称是孢子光产物，SP)。值得注意的是，孢子光产物的形成与细菌孢子对紫外线辐射的异常高抗性有关，这种抗性部分来自于修复SP的新型DNA修复酶。这项提案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，还描述了以环丁烷嘧啶二聚体为代价来探索SP形成机理的实验。本研究的具体目的如下：1)用适当的光谱方法研究孢子光产物裂解酶中铁硫簇的结构和环境；2)用同位素标记和底物类似物探索DNA修复反应的机理细节；3)用凝胶位移分析和DNA足迹法研究SPL与紫外线损伤DNA相互作用的分子基础；4)用穆斯堡尔谱、电子-核双共振和电子自旋回波包络调制研究SP裂解酶铁硫簇与底物S-腺苷蛋氨酸和孢子光产物的相互作用；(5)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.