Investigations Into Dynamic DNA Recognition and Processing During Eukaryotic Nucleotide Excision Repair

DNA受到内源性和环境源的持续损伤。未修复的DNA损伤会干扰复制和转录等细胞功能，通常会导致突变和基因组不稳定。核苷酸切除修复(NER)是一种主要的DNA修复途径，可以去除DNA中由紫外线、燃料燃烧、工业污染物、香烟烟雾等引起的结构多样化的损伤。NER的缺陷会导致超紫外线敏感性，并增加突变，从而导致人类包括癌症在内的疾病。本项目旨在获得对NER启动的关键步骤的详细的分子和结构了解。该项目的技术创新将适用于许多其他涉及复杂和动态蛋白质-DNA相互作用的系统。这项研究将为本科生和研究生研究人员提供跨学科培训机会，并通过与宾夕法尼亚大学医学院的密切合作，改善贝勒大学的生物医学研究环境。外展活动将包括参加贝勒的先进仪器研讨会，为当地学院/大学的教职员工和学生举办的高级仪器有限的机会；与得克萨斯州的一所高中合作开发基于研究的生物课程《基因组的守护者》，以及通过美国国家科学基金会资助的贝勒梅伯恩博物馆的公共门户计划进行公开演示。NER是一种基本的基因组维护机制，其分子机制在从酵母到人类的所有真核生物中都是保守的。虽然人们对NER的生化步骤知之甚少，但由于在合适的NER损伤上制备关键的多蛋白复合体(例如，TFIIH和Rad4(哺乳动物XPC的酵母同源物))用于结构研究的挑战，在结构水平上知之甚少。在该团队先前成功的基础上，通过结合冷冻电子显微镜(Cryo-EM)、交联质谱(XL-MS)、基于荧光寿命(Flt)的构象分析以及酵母遗传学，该项目旨在通过复杂结构及其转变的动态三维视图来全面了解NER的启动。具体地说，该项目将解决：(1)TFIIH-Rad4如何首先开始解开真正NER病变上的DNA，(2)如何通过分离转录特异的TFIIK激酶模块为NER激活holoTFIIH，以及(3)TFIIH-Rad4复合体如何过渡到病变验证阶段。这项使用酵母系统的研究还将为理解人类中各种NER连锁的表型打开新的大门，并阐明NER如何在活体中被调节。该项目由生物科学管理局分子和细胞生物科学部的遗传机制和分子生物学计划联合资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

DNA undergoes continuous damage from endogenous and environmental sources. Unrepaired DNA damage interferes with cellular functions such as replication and transcription, often resulting in mutations and genome instability. Nucleotide excision repair (NER) is a major DNA repair pathway that removes structurally diverse lesions caused by UV, fuel combustion, industrial pollutants, cigarette smoke, etc. in DNA. Defects in NER can cause hyper-UV sensitivity and increase mutations that lead to diseases including cancers in humans. This project aims to obtain detailed molecular and structural understanding of critical steps in the initiation of NER. Technical innovations in this project will be applicable to many other systems involving complex and dynamic protein-DNA interactions. The research will provide cross-disciplinary training opportunities for undergraduate and graduate researchers and enhance the biomedical research environment at Baylor University through close collaboration with the University of Pennsylvania medical school. Outreach activities will include participation in an Advanced Instrumentation Workshop at Baylor, held for faculty and students from local colleges/universities with limited access to high-level instrumentation; development of a research-based biology curriculum ‘Guardians of the Genome’ in collaboration with a high school in Texas, and public demonstrations through the NSF-funded ‘Portal to the Public’ program at Baylor’s Mayborn Museum.NER is an essential genome maintenance mechanism whose molecular machinery is conserved in all eukaryotes ranging from yeast to humans. Though much is known about the biochemical steps of NER, little is known at the structural level due to challenges in preparing key multiprotein complexes (e.g., TFIIH and Rad4 (yeast homolog of mammalian XPC)) on suitable NER lesions for structural studies. Building on the team’s prior successes and by combining cryo-electron microscopy (cryo-EM), cross-linking/mass spectrometry (XL-MS), and fluorescence lifetime (FLT)-based conformational analyses as well as yeast genetics, this project aims at comprehensive understanding of NER initiation through dynamic 3-D views of the complex structures and their transitions. Specifically, the project will address: (1) How TFIIH-Rad4 first starts unwinding the DNA on bona fide NER lesions, (2) How holoTFIIH is activated for NER by disengaging the transcription-specific TFIIK kinase module, and (3) How the TFIIH-Rad4 complex transitions to lesion verification stage. This research using the yeast system will also open new doors to understanding various NER-linked phenotypes in humans and shed light on how NER may be modulated in vivo.This project is jointly funded by the Genetic Mechanisms and Molecular Biophysics programs of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.