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

项目概要： DNA损伤加工和核苷酸切除修复启动的机制 本研究的目的是确定核苷酸切除修复的结构机制 (NER)入会仪式NER是最通用的DNA修复机制，可以修复各种各样的DNA损伤。 DNA损伤通过涉及30多种不同蛋白质的多步骤过程。必不可少的是， 在保持基因组完整性的同时，该途径也是从酵母到人类高度保守的。 NER因子的遗传缺陷导致从极端癌症易感性 综合征（着色性干皮病）至严重神经发育缺陷（Cockayne 综合征），从而提供了一个独特的范例，以了解不同的临床结果的DNA 损害最近的研究也揭示了NER作为体细胞突变热点的主要贡献者 在各种散发性癌症中，NER已被认为是抗癌的有吸引力的靶点 疗法 尽管它在生物学和医学上具有重要意义，但描述NER的机制一直是一个挑战。 由于净入学率因素的复杂组成和功能以及缺乏 对它们在DNA上相互作用的全面结构理解。该提案旨在定义 详细的三维结构中NER引发的机制，使用冷冻EM结合时间- 分辨荧光光谱和交联/质谱。结果会 回答关于（1）两个关键的NER启动器，Rad 4-Rad 23-Rad 33 （XPC-RAD 23 B-CETN 2的酵母同系物）和TFIIH一起启动DNA“打开”， 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 过程这种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，我们的研究将为一些本科生提供坚实的培训基础。 研究人员每年，并将显着提高生物医学研究环境， 贝勒大学，一个以本科生为重点的机构，通过其密切合作， 宾夕法尼亚大学医学院。沉浸在一个跨学科的研究项目与访问切割- 边缘技术，我们的本科研究人员将获得各种生物化学和 生物物理方法和成长为重要科学的关键驱动力。

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.