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: 10596096
• 负责人: Ivaylo Nikolaev Ivanov
• 金额: $ 41.16万
• 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/10596096
• 关键词: Acceleration; Aging; Alkylating Agents; Area; Binding Proteins; Biochemical; 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; 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; 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; Proliferating; Proteins; Pyrimidine Dimers; Reactive Oxygen Species; Regulation; Repair Complex; Resolution; Resources; Roentgen Rays; Role; Sampling; Scanning; Science; Single-Stranded DNA; 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.

项目概要/摘要 核苷酸切除修复（NER）是一种重要的基因组维护途径，可检测并去除 由于接触环境致癌物、毒素、烷化剂、反应性物质而导致有害 DNA 损伤 氧物质和紫外线辐射。 NER 在所有 DNA 修复途径中脱颖而出，因为它能够去除 最广泛的结构上不相关的病变。由于需要处理各种受损场地， 发展成为一个非常复杂的分子机器。该机器的缺陷为不同的 DNA 损伤的临床后果并与严重的人类疾病相关 – 1) 紫外线辐射 – 敏感综合症； 2) 着色性干皮病，具有极端的癌症倾向； 3) 脑- 眼面骨骼综合征； 4）毛发硫营养不良症； 5) Cockayne 综合征，与早产有关 衰老和加速的神经退行性变。此外，NER 与其他重要途径错综复杂地交织在一起 协调基因的表达和修复。因此，了解NER的分子机制是 生物医学领域的巨大挑战。规模和复杂性阻碍了实现这一目标的进展 以及完成 NER 的组件的动态性质。为了克服这一进步的关键障碍，我们 将采用综合建模方法，将最先进的计算与实验数据相结合 冷冻电子显微镜 (cryo-EM)、定点突变、交联质谱 (XL-MS)、 氢氘交换 (HDX) 质谱和小角 X 射线散射 (SAXS) 来阐明 关键 NER 复合物的组装、功能和调节。具体来说，我们的重点是转录因子 IIH (TFIIH) 作为 NER 机器的核心。在 Aim1 中，我们将阐明 TFIIH 的功能动力学 并发现关键的变构残基网络，使这个公认的 NER 主协调器的功能发挥作用。 我们还将破译 TFIIH 疾病突变的影响，为多样化的临床提供新的范例 NER损伤的表现。在 Aim2 中，我们将揭示 TFIIH 相关病变扫描的机制 和DNA损伤验证。在Aim3中，我们将综合不同的结构数据来创建一个综合模型 NER 中最关键的中间体——切开前复合体。混合模型将定义结构 使 TFIIH 能够作为 NER 机械组装和改造的移动平台。 我们的工作将受益于与世界一流实验小组的协同合作互动，以告知， 验证并扩展我们的模型。并行计算和实验的进步将产生重要的见解 NER 复合物的结构、动力学和功能，同时与遗传疾病直接相关 表型。因此，该项目的成功将对了解疾病病因和 提供一个结构框架来设计有效的治疗方法。