Integrative Modeling of Biomolecular Machinery in Nucleotide Excision Repair

项目摘要/摘要 核苷酸切除修复(NER)是一种重要的基因组维持途径，它检测和移除 接触环境致癌物质、毒素、烷化剂、反应性物质造成的有害DNA损伤 氧物种和紫外线辐射。在所有DNA修复途径中，NER因其移除 最广泛的结构上无关的病变。需要处理各种各样的损坏地点，这使得 上升到一个非常复杂的分子机制。这一机制的缺陷为不同的 DNA损伤的临床后果，并与严重的人类疾病有关-1)紫外线辐射- 敏感综合征；2)色素性干皮病，以极端癌症易感性为特征；3)大脑- 眼面部骨骼综合征；4)毛发硫代营养不良；5)Cockayne综合征，与早产有关 衰老和加速的神经退化。此外，NER与其他重要途径错综复杂地交织在一起 来协调基因的表达和修复。因此，了解NER的分子机制是 这是生物医学科学中的一项重大挑战。这一目标的进展一直受到规模和复杂性的阻碍 以及实现NER的组件的动态特性。为了克服这一取得进展的关键障碍，我们 将采用综合建模方法，将最先进的计算与来自 低温电子显微镜(Cryo-EM)、定点诱变、交联质谱(XL-MS)、 氢-氢交换(HDX)质谱学和小角X射线散射(SAXS)的研究 NER关键复合体的组装、功能和调控。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，从而实现该公认的NER主协调器的功能。 我们还将破译TFIIH疾病突变的影响，为不同的临床提供一个新的范例 NER损害的表现。在AIM2中，我们将揭开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中最关键的中间环节--切开前复合体。混合模式将定义结构 使TFIIH成为组装和改造NER机械的移动平台的要素。 我们的工作将受益于与世界级实验小组的协同协作互动，以告知， 验证并扩展我们的模型。并行的计算和实验进展将产生关键的见解 与遗传病直接相关的NER复合体的结构、动力学和功能 表型。因此，项目的成功将产生重大影响--无论是在理解疾病病因方面，还是在 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.