Unveiling how the XPC nucleotide excision repair complex senses DNA damage

细胞中的DNA不断受到各种来源的破坏，包括来自太阳的紫外线、工业污染物、香烟烟雾和烧焦的食物。这些DNA损伤如果不加以修复，可能会阻碍重要的细胞功能，并在遗传密码中产生错误，从而可能导致细胞死亡或疾病。该项目将解决DNA修复领域的两个关键问题：修复酶如何在正常DNA的巨大“基因组海洋”中检测少量DNA损伤？为什么有些损伤修复得非常有效，而有些则完全不能修复？利用创新的、多学科的方法，该研究将以前所未有的细节将当前病变检测过程的静态视图转变为三维分子轨迹“电影”，从而提供重要的见解，最终可能导致开发更好的方法来防止DNA损伤诱导的细胞缺陷的发生。同样重要的好处将来自于将研究整合到创造性教育和推广项目中，以促进研究生和本科生在化学和物理方面的跨学科培训，并建立教育模块，结合新的下一代科学标准的横断面概念，供芝加哥公立学校的K-12教师和学生使用。XPC复合体识别来自基因组DNA的多种环境诱导的DNA损伤，因此是真核生物核苷酸切除修复途径启动的关键。病变的识别效率因病变的不同而有很大差异，某些病变可以逃避XPC的检测，从而对核苷酸切除修复产生抗性。该研究小组先前的研究提出了一种新的“动力学门控”机制，其中XPC区分病变和正常位点的能力可能在于形成“开放”构象的动力学，受损（或正常）核苷酸被翻转出来。该项目的目标是通过x射线晶体学，时间分辨温度跳变荧光光谱和化学交联等互补技术的独特组合来严格研究该模型。具体目的是：(1)表征和比较XPC诱导的受损和正常DNA打开的动力学，(2)利用缺乏关键DNA结合结构元件的突变XPC确定病变识别的动力学和结构，以及(3)阐明和比较XPC与修复抗性和熟练病变结合的结构和动力学。该结果将为病变识别动力学提供新的见解。此外，本研究中开发的独特方法也将与其他基因调控和维持系统相关，并可能有助于揭示由瞬态相互作用控制的蛋白质功能的新范式，这是以前通过其他方法无法检测到的。

DNA in cells is continuously being damaged by diverse sources including UV from sun, industrial pollutant, cigarette smoke, and burnt food. These DNA lesions, if left unrepaired, can block important cellular functions and create errors in the genetic code, which may lead to cell death or diseases. This project will tackle two key questions in the field of DNA repair: How do repair enzymes detect a small number of DNA lesions in a huge "genomic ocean" of normal DNA? Why are some lesions repaired very efficiently, whereas others are not repaired at all? Using innovative, multi-disciplinary approaches, the research will transform the current static view of the lesion detection process into a 3-D molecular trajectory "movie" in unprecedented detail, thus providing important insights that might eventually lead to development of better ways to prevent the occurrence of DNA damaged-induced cellular defects. Equally important benefits will come from Integration of the research into creative education and outreach programs to promote cross-disciplinary training in Chemistry and Physics for graduate and undergraduate students and to build educational modules, incorporating crosscutting concepts of the new Next Generation Science Standards, to be used by K-12 teachers and their students in the Chicago Public Schools.The XPC complex recognizes diverse, environmentally induced DNA lesions from the genomic DNA, and thus is a key to the initiation of the eukaryotic nucleotide excision repair pathway. The recognition efficiency of the lesions can vary widely depending on the lesion, and certain lesions can evade detection by XPC and thus become resistant to nucleotide excision repair. Previous studies from this research group have suggested a novel "kinetic gating" mechanism in which XPC's ability to discriminate between lesions and normal sites may lie in the kinetics of forming an "open" conformation with the damaged (or normal) nucleotides flipped out. The goal of this project is to rigorously investigate this model by a unique combination of complementary technologies including X-ray crystallography, time-resolved temperature-jump fluorescence spectroscopy and chemical crosslinking. Specific aims are (1) to characterize and compare the dynamics of XPC-induced DNA opening in damaged and normal DNA, (2) to determine the dynamics and structures of lesion recognition using mutant XPC that lack key DNA-binding structural elements, and (3) to elucidate and compare the structure and kinetics of XPC's binding to repair-resistant and -proficient lesions. The results will provide new insights into the dynamics of lesion recognition. Moreover, the unique approaches developed in this study will also be relevant to other gene regulatory and maintenance systems, and may help uncover a new paradigm of protein functions controlled by transient interactions, which have escaped previous detection by other methods.