Mechanisms of DNA damage processing and the initiation of Nucleotide Excision Repair

项目总结： DNA损伤处理机制与核苷酸切除修复的启动 本研究的目的是确定核苷酸切除修复的结构机制。 (NER)入会。NER是最通用的DNA修复机制，可以修复各种各样的 DNA损伤通过一个涉及30多种不同蛋白质的多步骤过程。是必不可少的 为了保持基因组的完整性，这条途径从酵母到人类也是高度保守的。 NER因子的遗传缺陷导致从极端癌症易感性到表型的各种表型 综合征(着色性干皮病)到严重的神经发育缺陷(Cockayne 综合症)，从而提供了一种独特的范式来理解DNA的不同临床结果 损坏。最近的研究还表明，NER是体细胞突变热点的主要贡献者 在各种散发性癌症中，NER已被认为是一个有吸引力的抗癌靶点 心理治疗。 尽管NER具有生物学和医学上的重要性，但阐明NER的机制一直是一个 由于NER因子的复杂组成和功能以及缺乏而带来的长期挑战 对它们在DNA上的相互作用的全面结构理解。这项提案旨在界定 利用低温电磁与时间-时间相结合的方法研究了NER在详细三维结构中的起爆机理。 分辨荧光光谱和交联质谱联用。结果将是 回答有关以下方面的基本问题：(1)两个关键的NER发起者RAD4-RAD23-RAD33如何 (XPC-RAD23B-CETN2的酵母同源物)和TFIIH一起启动DNA在 损伤-NER启动的关键步骤，以及(2)DNA中的扭转应力如何影响这一点 进程。这一理解将为解释各种病理生理学提供基础。 涉及NER，这反过来又可以导致对抗各种NER相关疾病的新策略 包括癌症。 重要的是，我们的研究将为几位本科生提供坚实的培训基础 每年研究人员，并将显著改善生物医学研究环境 贝勒大学，一所专注于本科生的机构，通过与 宾夕法尼亚大学医学院。沉浸在一个跨学科的研究项目中，可以接触到- 尖端技术，我们的本科生研究人员将获得各种生化和 生物物理方法，并作为重大科学的关键驱动力而增长。

Project Summary: Mechanisms of DNA damage processing and the initiation of Nucleotide Excision Repair The goal of this research is to determine the structural mechanism of nucleotide excision repair (NER) initiation. NER is the most versatile DNA repair mechanism that repairs a wide variety of DNA lesions through a multistep process involving over 30 different proteins. Being essential to maintaining genome integrity, this pathway is also highly conserved from yeast to humans. Genetic defects in NER factors lead to phenotypes ranging from extreme cancer predisposition syndrome (xeroderma pigmentosum) to severe neurodevelopmental defects (Cockayne syndrome), thus providing a unique paradigm to understand diverse clinical outcomes of DNA damage. Recent studies also revealed NER as a major contributor of somatic mutation hotspots in various sporadic cancers and NER has been suggested as an attractive target for anti-cancer therapy. Despite its biological and medical importance, delineating the mechanisms of NER has been a long-term challenge due to the complex compositions and functions of NER factors and the lack of comprehensive structural understanding of their interplay on DNA. This proposal aims to define the mechanism of NER initiation in detailed 3D structures using cryo-EM combined with time- resolved fluorescence spectroscopy and crosslinking/mass-spectrometry. The outcome will answer fundamental questions regarding (1) how the two key NER initiators, Rad4-Rad23-Rad33 (yeast homolog of XPC-RAD23B-CETN2) and TFIIH, together start the DNA ‘opening’ around the damage - a critical step in NER initiation, and (2) how the torsional stress in DNA impacts this process. This understanding will provide the foundation to explain various pathophysiologies involving NER, which in turn can lead to novel strategies to counter various NER-linked diseases including cancer. Importantly, our research will provide solid training grounds for several undergraduate researchers every year and will significantly enhance the biomedical research environment at Baylor University, an undergraduate-focused institution, through its intimate collaboration with UPenn Medical School. Immersed in an interdisciplinary research project with access to cutting- edge technologies, our undergraduate researchers will gain expertise in various biochemical and biophysical approaches and grow as key drivers of significant science.