Structural determination of DNA damage identification by the repair protein XPC

修复蛋白 XPC 鉴定 DNA 损伤的结构测定

• 批准号: 7908543
• 负责人: Steven M. Shell
• 金额: $ 5.05万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7908543
• 关键词: Affect; Affinity; Binding; Biochemical; Carcinogens; Cells; Chemicals; Complex; DNA; DNA Binding; DNA Damage; DNA lesion; Development; Environment; Escherichia coli; Exposure to; Fluorescence Anisotropy; Genome; Goals; Human; Induced Mutation; Instruction; Lead; Length; Lesion; Malignant Neoplasms; Mutation; Nucleotide Excision Repair; Prevention therapy; Primary Lesion; Process; Property; Proteins; Site; Structure; Testing; Ultraviolet Rays; X-Ray Crystallography; cancer prevention; cigarette smoking; design; ds-DNA; improved; insight; preference; prevent; public health relevance; reconstitution; repaired; structural biology

DESCRIPTION (provided by applicant): The instructions necessary for the proper development and function of each cell are encoded in the DNA the cell carries. However, DNA is susceptible to damage from toxic agents found in the environment. DNA damage can cause mutation, changes in the cellular instructions, which can lead to the development of cancer in humans. Nucleotide excision repair (NER) is a process that identifies and removes DNA lesions to prevent mutation. NER has the remarkable ability to identify many types of unrelated lesions, although this property is poorly understood. XPC is the primary lesion detecting protein in global genome NER (GG-NER). XPC binds to both single-stranded and double-stranded DNA but displays a preference for damaged duplex DNA. Determining how XPC interacts with DNA is critical for understanding how exposure to carcinogens, such as ultraviolet light and chemical commonly found in cigarette smoke, lead to cancer. In this proposal I will investigate the biochemical and structural interactions between XPC and DNA to determine how DNA lesions are identified in GG-NER. The hypothesis of this proposal is that XPC will bind any DNA that deviates from the normal structure but binds DNA containing bulky chemical lesions tighter. Two specific aims are designed to test this hypothesis. Aim 1 will use fluorescence anisotropy to determine the affinity of the XPC for DNA substrates containing different types of lesions to test whether the chemical identity of the lesion affects the ability of XPC to identify DNA damage sites. Aim 2 will elucidate the molecular interactions underlying DNA damage recognition in NER by determining the structure of XPC-damaged DNA complex by X-ray crystallography. In order to achieve these goals, the DNA binding apparatus of human XPC (XPCDNA) has been isolated and characterized. The XPCDNA construct reconstitutes the DNA binding properties of the full-length protein and maintains the ability to discriminate between damaged and undamaged DNA. However, the XPCDNA protein can be purified from E. coli in sufficient quantity and purity for structural biology studies. PUBLIC HEALTH RELEVANCE: This study will provide valuable new insight into how DNA damage is recognized in NER. Determining how DNA damage is detected will greatly improve our understanding of how mutation induces cancer in humans. Ultimately understanding how DNA damage is recognized has the potential to lead to advances in both cancer prevention and therapy.

描述(由申请人提供)：每个细胞正常发育和功能所需的指令编码在细胞携带的DNA中。然而，DNA很容易受到环境中发现的有毒物质的破坏。DNA损伤会导致突变，细胞指令的变化，这可能会导致人类癌症的发展。核苷酸切除修复(NER)是识别和移除DNA损伤以防止突变的过程。NER具有识别许多类型的无关病变的非凡能力，尽管人们对这一特性知之甚少。XPC是全球基因组NER(GG-NER)中主要的病变检测蛋白。XPC既能与单链DNA结合，又能与双链DNA结合，但偏爱受损的双链DNA。确定XPC与DNA的相互作用对于了解接触致癌物质(如紫外线和香烟烟雾中常见的化学物质)如何导致癌症至关重要。在这项建议中，我将研究XPC和DNA之间的生化和结构相互作用，以确定DNA损伤是如何在GG-NER中识别的。这一提议的假设是，XPC将结合任何偏离正常结构的DNA，但会更紧密地结合包含巨大化学损伤的DNA。我们设计了两个具体的目标来检验这一假设。目的1将利用荧光各向异性来确定XPC与含有不同类型病变的DNA底物的亲和力，以测试病变的化学特性是否影响XPC识别DNA损伤位置的能力。目的2通过X射线结晶学测定XPC损伤DNA复合体的结构，阐明NER中DNA损伤识别的分子相互作用。为了实现这些目标，人们已经分离并鉴定了人XPC的DNA结合装置(XPCDNA)。XPCDNA结构重组了全长蛋白质的DNA结合特性，并保持了区分受损和未受损DNA的能力。然而，XPCDNA蛋白可以从大肠杆菌中纯化出足够数量和纯度的蛋白，用于结构生物学研究。 公共卫生相关性：这项研究将为NER如何识别DNA损伤提供有价值的新见解。确定DNA损伤是如何被检测到的，将极大地提高我们对突变如何导致人类癌症的理解。最终，了解DNA损伤是如何识别的，有可能导致癌症预防和治疗的进步。