Integrative Modeling of Biomolecular Machinery in Nucleotide Excision Repair

项目总结/摘要 核苷酸切除修复（NER）是一种重要的基因组维护途径， 由于暴露于环境致癌物、毒素、烷化剂、反应性 氧物种和紫外线辐射。NER在所有DNA修复途径中脱颖而出，因为它能够去除 最广泛的结构无关的病变由于需要处理各种各样的受损地点， 形成了非常复杂的分子结构这一机制的缺陷为不同的人提供了一个范例。 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- 敏感综合征; 2）着色性干皮病，以极端癌症易感性为特征; 3）脑- 眼-面-骨骼综合征; 4）脑硫营养不良;和5）Cockayne综合征，与早产儿相关 老化和加速的神经退化。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 这是生物医学科学的一个重大挑战。实现这一目标的进展一直受到规模、复杂性和 和实现NER的组件的动态性质。为了克服这一重大障碍，我们 将采用综合建模方法，结合最先进的计算与实验数据， 冷冻电子显微镜（cryo-EM）、定点诱变、交联质谱（XL-MS）， 氢氘交换（HDX）质谱和小角X射线散射（SAXS），以阐明 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机制的核心。在目标1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。 我们还将破译TFIIH疾病突变的影响，为不同的临床研究提供新的范例。 净入学率受损的表现。在目标2中，我们将阐明TFIIH相关病变扫描的机制 和DNA损伤鉴定在Aim 3中，我们将综合各种结构数据以创建一个集成模型 NER中最关键的中间体--切口前复合体。混合模型将定义结构 这些元素使TFIIH能够作为一个移动的平台，用于装配和改造NER机器。 我们的工作将受益于与世界级实验小组的协同合作互动， 验证和扩展我们的模型。并行计算和实验的进步将产生关键的见解， 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，该项目的成功将产生重大影响-无论是在了解疾病的病因， 提供了一个结构框架来设计有效的治疗方法。

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.