STRUCTURE, FUNCTION AND EVOLUTION OF DNA REPAIR ENZYMES

描述（由申请方提供）：羟基自由基在内源性和暴露于电离辐射后形成，并产生广谱的潜在致死性和致突变性DNA损伤，这些损伤可通过碱基切除修复（BER）酶清除。BER已被广泛研究使用裸DNA底物，但几乎没有直接的信息BER酶如何处理自由基诱导的DNA损伤的核小体，这是染色质的基本亚基。提出的研究的双重目标是确定核小体中氧化损伤的识别和修复中的限速步骤，并确定调节或规避这些限速步骤的因素。为此，我们将组装模型核小体，包含离散定位，定义的病变或DNA结构，代表BER途径中的中间体。这些将与纯化的人双功能DNA糖基化酶和选择的其他BER酶一起孵育，以测试假设1，即核小体中自由基诱导的单碱基DNA损伤的BER中的限速步骤是损伤识别步骤，其由DNA损伤相对于核小体二分体的旋转和平移位置控制。然后，我们将检验假说2，即核小体结构的改变允许BER糖基化酶作用于空间上无法接近的病变。我们将研究组蛋白八聚体的DNA部分可逆解包在多大程度上有助于进入空间闭塞的病变。含有病变的核小体也将与酵母核提取物一起孵育，所述酵母核提取物由野生型细胞和在选定的BER辅助因子或染色质重塑剂中有缺陷的突变体制备，以帮助指导未来分离调节细胞中BER的因子的努力。 与该项目的其他组成部分的整合：该研究计划中目前资助的项目集中在DNA修复酶的三个家族的结构，酶的性质和底物偏好，其中两个在本提案中概述的研究中占有突出地位。此外，这些研究将大大拓宽我们对人类DNA糖基化酶如何与核小体中的病变相互作用的理解，并将潜在地揭示增强染色质中病变BER的因素。除了这些共同的主题目标，这些研究将从生物信息学和酶纯化服务中受益，这些服务将通过项目项目内的核心A和B提供。 与公共卫生的关系：拟议的研究所产生的信息将促进我们对辐射暴露导致癌症发展的基本机制的理解。此外，放射疗法广泛用于癌症治疗。这些研究将促进我们对影响细胞辐射敏感性的因素的理解。这些因素的调制可以帮助最大限度地提高放疗期间的治疗增益。

DESCRIPTION (provided by the applicant): Hydroxyl radicals form endogenously and following exposure to ionizing radiation, and produce a broad spectrum of potentially lethal and mutagenic DNA lesions that are removed by Base Excision Repair (BER) enzymes. BER has been studied extensively using naked DNA substrates, but there is virtually no direct information on how BER enzymes process free radical-induced DNA damage in nucleosomes, which are the fundamental subunit of chromatin. The twin goals of the proposed studies are to identify rate-limiting steps in the recognition and repair of oxidative lesions in nucleosomes, and to identify factors that regulate or circumvent these rate-limiting steps. To that end, we will assemble model nucleosomes that contain discretely positioned, defined lesions or DNA structures that represent intermediates in the BER pathway. These will be incubated with purified, human bifunctional DNA glycosylases and selected other BER enzymes to test HYPOTHESIS 1, that the rate-limiting step in BER of free radical-induced single base DNA lesions in nucleosomes is the lesion recognition step, which is governed by the rotational and translational position of DNA damage relative to the nucleosome dyad. We then will test HYPOTHESIS 2, that alterations in nucleosome structure permit BER glycosylases to act on lesions that would otherwise be sterically inaccessible. We will investigate the extent to which partial, reversible unwrapping of DNA from the histone octamer facilitates access to sterically occluded lesions. Lesion-containing nucleosomes will also be incubated with yeast nuclear extracts, prepared from wild type cells and mutants defective in selected BER accessory factors or chromatin remodeling agents to help guide future efforts to isolate factors that regulate BER in cells. INTEGRATION WITH OTHER COMPONENTS OF THE PROGRAM PROJECT: The currently funded projects in this Research Program focus on the structure, enzymatic properties, and substrate preferences of three families of DNA repair enzymes, two of which figure prominently in studies outlined in this proposal. Moreover these studies will substantially broaden our understanding of how human DNA glycosylases interact with lesions in nucleosomes, and will potentially reveal factors that enhance BER of lesions in chromatin. In addition to these common thematic goals, these studies will materially benefit from the bioinformatics and enzyme purification services that will be available through Cores A and B within the program project. RELEVANCE TO PUBLIC HEALTH: The information generated by the proposed studies will advance our understanding of the basic mechanisms that lead from radiation exposure to development of cancer. Additionally, radiation therapy is widely used in cancer therapy. The proposed studies will advance our understanding of factors that affect radiation sensitivity of cells. Modulation of such factors could help maximize therapeutic gain during radiotherapy.