The XPA scaffold protein in Nucleotide Excision Repair

核苷酸切除修复中的 XPA 支架蛋白

• 批准号: 10334466
• 负责人: WALTER J. CHAZIN
• 金额: $ 31.24万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-09 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10334466
• 关键词: Address; Affect; Antineoplastic Agents; Area; Binding; Biochemical; Biochemistry; C-terminal; Cancer Etiology; Carboplatin; Cells; Cellular Assay; Cellular biology; Cessation of life; Chemistry; Cisplatin; Complex; DNA; DNA Adducts; DNA Damage; DNA Repair; DNA Repair Disorder; DNA Repair Pathway; DNA lesion; DNA-Protein Interaction; Defect; Drug Prescriptions; Ensure; Evaluation; Excision; Foundations; Gene Mutation; Genetic; Genome; Hereditary Disease; Human; In Vitro; Incidence; Individual; Laboratories; Lead; Lesion; Mediating; Molecular; Mutagens; Mutation; Neurologic; Nucleotide Excision Repair; Oligonucleotides; Oncology; Pathway interactions; Pharmaceutical Preparations; Phenotype; Positioning Attribute; Predisposition; Property; Proteins; RAD23B gene; Radiation; Reaction; Repair Complex; Resistance; Resistance development; Role; Scaffolding Protein; Site; Skin Cancer; Source; Structure; Sunlight; Surface; Surgical incisions; Testing; Toxic Environmental Substances; Transcription-Coupled Repair; Work; XPA gene; Xeroderma Pigmentosum; anticancer research; antitumor agent; base; biophysical techniques; cancer therapy; gene repair; improved; in vivo; inhibitor; insight; interest; multidisciplinary; premature; protein protein interaction; recruit; repaired; scaffold; small molecule; small molecule inhibitor; solar ultraviolet radiation; structural biology; success; therapy outcome; transcription factor TFIIH; tumor; ultraviolet damage; ultraviolet irradiation; xeroderma pigmentosum group A complementing protein

Nucleotide excision repair (NER) protects human cells by removing harmful DNA adducts formed by environmental toxins and solar UV irradiation. Defects in NER in humans lead to the DNA repair disorder xeroderma pigmentosum, which is characterized by high predisposition to skin cancer, neurological abnormalities and premature death. The repair of damaged DNA by NER also has a downside – it contributes significantly to the development of resistance of tumors to treatment with antitumor agents, in particular cis- and carboplatin, two of the most widely prescribed drugs in oncology. We propose to leverage the combined expertise of the Scharer and Chazin laboratories in chemistry, biochemistry, cell biology, structural biology, and small molecule discovery to elucidate how the scaffold protein XPA coordinates the assembly and organization of NER incision complexes. Despite its modest size (273 residues), XPA functions as the central scaffold of NER complexes, interacting with 4 key NER proteins as well as DNA. However, how XPA is recruited to the site of damage and positions other factors through its various interactions remains poorly understood. Moreover, given its essential role in organizing and orchestrating the trajectory of NER complexes, XPA is an attractive potential Achilles Heel to target for suppressing NER. Aim 1 will test the hypothesis that TFIIH recruits XPA to sites of UV damage through a proposed interaction interface involving the C-terminal region of XPA and the p8 subunit of TFIIH. We will biochemically and structurally characterize this interaction and determine how mutations in the interface that disturb this interaction affect NER in vitro and in vivo. Abolishing the interaction between XPA and TFIIH will also address the long-standing question of whether the steps following damage recognition are the same for global genome and transcription-coupled NER – the arrival of TFIIH and XPA is the first step common to both pathways. The action of TFIIH on damaged DNA creates an open “NER bubble” that provides a landing platform for XPA and RPA and is required for NER incision. The scaffolding function of XPA is reliant on its coordination with RPA, which binds the undamaged strand. Aim 2 will determine the molecular basis and functional implications of the coordinated action of XPA and RPA. Structural, biochemical, and biophysical approaches combined with cellular assays will test the hypothesis that the two interaction sites between XPA and RPA are simultaneously engaged and contribute to NER in a cooperative fashion. Based on these results, our expertise in fragment based molecular discovery will be used to develop and validate initial inhibitors targeting XPA-RPA interfaces. These studies are expected to provide: (i) dramatic new mechanistic insights into the central role of XPA in assembling and coordinating the NER machinery; (ii) the identity of XPA interaction surfaces that are critical to NER; (iii) proof of principle that interfaces between XPA and other NER factors are suitable targets for evaluating the potential of overcoming tumor resistance to DNA damaging therapies by suppressing NER.

核苷酸切除修复 (NER) 通过去除由以下物质形成的有害 DNA 加合物来保护人体细胞： 环境毒素和太阳紫外线照射导致人类 NER 缺陷导致 DNA 修复障碍。 色素性干皮病，其特征是高度易患皮肤癌、神经系统癌症 NER 修复受损 DNA 也有一个缺点——它会导致异常和过早死亡。 显着影响肿瘤对抗肿瘤药物治疗的耐药性的发展，特别是顺式- 和卡铂，这是肿瘤学中最广泛使用的两种药物。 我们建议利用 Scharer 和 Chazin 实验室在化学方面的综合专业知识， 生物化学、细胞生物学、结构生物学和小分子发现，以阐明支架蛋白如何 XPA 协调 NER 切口复合物的组装和组织，尽管其尺寸不大（273）。 残基），XPA 充当 NER 复合物的中心支架，还与 4 个关键 NER 蛋白相互作用 然而，XPA 如何被招募到损伤部位并通过其各种因素定位其他因素。 此外，鉴于其在组织和协调方面的重要作用，人们对其的了解仍知之甚少。 根据 NER 复合物的轨迹，XPA 是抑制 NER 的一个有吸引力的潜在致命弱点。 目标 1 将测试 TFIIH 通过拟议的相互作用将 XPA 招募到紫外线损伤部位的假设 涉及 XPA 的 C 末端区域和 TFIIH 的 p8 亚基的界面我们将进行生化和分析。 从结构上表征这种相互作用，并确定界面中的突变如何干扰这种相互作用 相互作用影响 NER 体外和体内 消除 XPA 和 TFIIH 之间的相互作用也将解决。 长期存在的问题是，损伤识别后的步骤对于全球基因组是否相同 和转录偶联的 NER——TFIIH 和 XPA 的到来是这两条途径共同的第一步。 TFIIH 对受损 DNA 的作用创造了一个开放的“NER 气泡”，为 XPA 提供了着陆平台 XPA 的支架功能依赖于其与 RPA 的协调。 RPA 结合未受损的链，目标 2 将确定分子基础和功能含义。 XPA 和 RPA 的协调作用与结构、生物化学和生物物理方法相结合。 细胞分析将检验 XPA 和 RPA 之间的两个相互作用位点同时存在的假设 基于这些结果，我们的专业知识碎片化。 基于分子发现的方法将用于开发和验证针对 XPA-RPA 界面的初始抑制剂。 这些研究预计将提供：（i）对 XPA 在 组装和协调 NER 机制；(ii) 对于 XPA 交互表面至关重要 NER；(iii) XPA 和其他 NER 因素之间的接口是合适目标的原理证明 评估通过抑制 NER 克服肿瘤对 DNA 损伤疗法耐药性的潜力。