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均编码在细胞携带的 DNA 中。然而，DNA 很容易受到环境中有毒物质的损害。 DNA 损伤会导致突变，细胞指令发生变化，从而导致人类癌症的发生。核苷酸切除修复 (NER) 是一个识别并去除 DNA 损伤以防止突变的过程。 NER 具有识别多种类型的不相关病变的非凡能力，尽管人们对这一特性知之甚少。 XPC 是全基因组 NER (GG-NER) 中的主要病变检测蛋白。 XPC 可结合单链和双链 DNA，但显示出对受损双链 DNA 的偏好。确定 XPC 如何与 DNA 相互作用对于了解暴露于致癌物质（例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。在本提案中，我将研究 XPC 和 DNA 之间的生化和结构相互作用，以确定如何在 GG-NER 中识别 DNA 损伤。该提案的假设是，XPC 会结合任何偏离正常结构的 DNA，但会更紧密地结合含有大体积化学损伤的 DNA。设计了两个具体目标来检验这一假设。目标 1 将利用荧光各向异性来确定 XPC 对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 损伤位点的能力。目标 2 将通过 X 射线晶体学确定 XPC 损伤的 DNA 复合物的结构，从而阐明 NER 中 DNA 损伤识别背后的分子相互作用。为了实现这些目标，人类 XPC 的 DNA 结合装置 (XPCDNA) 已被分离和表征。 XPCDNA 构建体重建了全长蛋白质的 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，其数量和纯度足以用于结构生物学研究。 公共健康相关性：这项研究将为 NER 中如何识别 DNA 损伤提供有价值的新见解。确定如何检测 DNA 损伤将极大地提高我们对突变如何诱发人类癌症的理解。最终了解 DNA 损伤如何被识别有可能推动癌症预防和治疗的进步。