Integrative Modeling of Biomolecular Machinery in Nucleotide Excision Repair

核苷酸切除修复中生物分子机械的集成建模

• 批准号: 10362051
• 负责人: Ivaylo Nikolaev Ivanov
• 金额: $ 44.04万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-24 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10362051
• 关键词: Aging; Alkylating Agents; Area; Binding Proteins; Biochemical; Biochemical Genetics; Carcinogen exposure; Cell Death; Cells; Cerebrooculofacioskeletal Syndrome; Chemicals; Cisplatin; Clinical; Cockayne Syndrome; Complex; Coupled; Cryoelectron Microscopy; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA lesion; Data; Defect; Detection; Deuterium; Development; Disease; ERCC3 gene; Elements; Endogenous Factors; Environmental Carcinogens; Environmental Health; Environmental Risk Factor; Epitopes; Equilibrium; Etiology; Evolution; Excision; Exposure to; Foundations; Free Energy; Genetic; Genetic Diseases; Genetic Transcription; Genome; Genomic DNA; Goals; Health; Heterogeneity; Human; Hybrids; Hydrogen; Impairment; Individual; Investigation; Knowledge; Lesion; Letters; Light; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Modeling; Modification; Molecular; Molecular Conformation; Motion; Mutation; Nature; Nerve Degeneration; Normal Range; Nucleotide Excision Repair; Outcome; Pathway Analysis; Pathway interactions; Patients; Phenotype; Platinum adduct; Positioning Attribute; Predisposition; Premature aging syndrome; Process; Proteins; Pyrimidine Dimers; Reactive Oxygen Species; Regulation; Repair Complex; Resolution; Resources; Roentgen Rays; Role; Sampling; Scanning; Science; Site; Site-Directed Mutagenesis; Structure; Supercomputing; Surgical incisions; Syndrome; Techniques; Toxin; Trichothiodystrophy; Ultraviolet Rays; Work; XPA gene; Xeroderma Pigmentosum; adduct; assault; cancer predisposition; crosslink; cytotoxic; disease phenotype; diverse data; ds-DNA; effective therapy; experience; experimental group; experimental study; gene repair; helicase; human disease; insight; mobile computing; novel; nuclease; parallel computer; protein complex; repaired; structural biology; success; transcription factor TFIIH

PROJECT SUMMARY/ABSTRACT Nucleotide excision repair (NER) is an essential genome maintenance pathway that detects and removes harmful DNA lesions resulting from exposure to environmental carcinogens, toxins, alkylating agents, reactive oxygen species and ultraviolet radiation. NER stands out among all DNA repair pathways for its ability to remove the widest array of structurally unrelated lesions. The need to process a wide variety of damaged sites has given rise to a remarkably complex molecular machinery. Defects in this machinery provide a paradigm for the diverse clinical consequences of DNA damage and are associated with severe human diseases – 1) ultraviolet radiation- sensitive syndrome; 2) xeroderma pigmentosum, characterized with extreme cancer predisposition; 3) cerebro- oculo-facio-skeletal syndrome; 4) trichothiodystrophy; and 5) Cockayne syndrome, associated with premature ageing and accelerated neurodegeneration. Furthermore, NER is intricately intertwined with other vital pathways that orchestrate the expression and repair of genes. Thus, understanding the molecular mechanisms of NER is a grand challenge in biomedical science. Progress toward this goal has been hindered by the size, complexity and dynamic nature of the assemblies that accomplish NER. To overcome this critical barrier to progress, we will employ integrative modeling methods, combining state-of-the-art computation with experimental data from cryo-electron microscopy (cryo-EM), site-directed mutagenesis, crosslinking mass spectrometry (XL-MS), hydrogen deuterium exchange (HDX) mass spectrometry and small angle X-ray scattering (SAXS) to elucidate the assembly, function and regulation of key NER complexes. Specifically, our focus is on transcription factor IIH (TFIIH) as the centerpiece of the NER machinery. In Aim1, we will elucidate the functional dynamics of TFIIH and discover key allosteric residue networks enabling the function of this recognized NER master coordinator. We will also decipher the effects of TFIIH disease mutations, providing a novel paradigm for the diverse clinical manifestations of NER impairment. In Aim2, we will unravel the mechanisms of TFIIH-associated lesion scanning and DNA damage verification. In Aim3, we will synthesize diverse structural data to create an integrative model of the most crucial intermediate in NER – the pre-incision complex. Hybrid models will define the structural elements allowing TFIIH to serve as a mobile platform for the assembly and remodeling of the NER machinery. Our work will benefit from synergistic collaborative interactions with world-class experimental groups to inform, validate, and extend our models. Parallel computational and experimental advances will yield key insights into the structure, dynamics and function of NER complexes while making direct connection to genetic disease phenotypes. Success of the project will thus have major impacts - both in understanding disease etiology and in offering a structural framework to devise effective treatments.

项目摘要/摘要 核苷酸惊喜修复（NER）是一种必不可少的基因组维持途径，可检测和去除 暴露于环境致癌物，毒素，烷基化剂，反应性导致的有害DNA水平 氧和紫外线辐射。 NER在所有DNA修复途径中脱颖而出，其能力去除 结构无关的病变最宽。需要处理各种损坏的站点的需求 上升到非常复杂的分子机械。该机械中的缺陷为潜水员提供了范式 DNA损伤的临床后果，与严重的人类疾病有关 - 1）紫外线辐射 - 敏感综合征； 2）甲状腺素色素，以极端癌症的易感性为特征； 3）小脑 - Oculo-facio-Skeletal综合征； 4）毛刺肌营养； 5）Cockayne综合征，与早产相关 衰老和加速神经变性。此外，NER与其他重要途径复杂地交织在一起 策划了基因的表达和修复。那是理解NER的分子机制 生物医学科学的巨大挑战。大小，复杂性阻碍了朝着这个目标朝着这个目标的进步 以及完成的组件的动态性质。为了克服这个关键的进步障碍，我们 将采用集成的建模方法，将最新计算与实验数据结合 冷冻电子显微镜（冷冻 - EM），位置定向诱变，交联质谱（XL-MS），XL-MS） 氢氘交换（HDX）质谱和小角度X射线散射（SAX）以阐明 关键NER复合物的组装，功能和调节。具体而言，我们的重点是转录因子 IIH（TFIIH）是NER机械的核心。在AIM1中，我们将阐明TFIIH的功能动力学 并发现关键的变构居住网络，以实现该公认的NER主协调员的功能。 我们还将破译TFIIH疾病突变的影响，为潜水员临床提供新的范式 NER损害的表现。在AIM2中，我们将阐明与TFIIH相关病变扫描的机制 和DNA损伤验证。在AIM3中，我们将合成潜水的结构数据以创建一个集成模型 NER中最关键的中间体 - 结构前复合物。混合模型将定义结构 允许TFIIH充当NER机械组装和重塑的移动平台的元素。 我们的工作将受益于与世界一流的实验小组的协同协作互动，以告知， 验证并扩展我们的模型。并行计算和实验进步将产生关键的见解 NER复合物的结构，动力和功能，同时直接与遗传疾病联系 表型。因此，该项目的成功将对疾病病因和在内产生重大影响 提供一个结构框架来设计有效的治疗方法。