Molecular Mechanisms for DNA Damage Processing by Transcription Machinery

项目摘要/摘要 这个项目的长期目标是了解DNA损伤是如何在 活跃转录的基因组。由内源性和环境因素引起的有害DNA损伤必须 及时识别和修复，以避免对基因组完整性的有害威胁。转录- 偶联核苷酸切除修复(TC-NER)是去除DNA损伤的一条重要的DNA修复途径 在转录后的基因组中。然而，对于真核细胞中TC-NER的分子机制却知之甚少。 入会仪式。Cockayne综合征B蛋白(CSB)是TC-NER的主要协调者，被招募到DNA损伤- 在真核细胞TC-NER的启动过程中起着关键作用。此前，我们报道了第一个 酵母Pol II-Rad26/CSB三元复合结构，揭示了这一重要过程的新亮点。然而， 在理解公务员事务局招募到DNA后会发生什么方面，仍然存在基本的知识差距 病变被逮捕POL II。该领域的几个长期存在的问题仍未得到回答。首先，公务员事务局如何 利用其DNA转位酶活性重塑DNA损伤受阻的POL II，并将POL II从 转录延伸模式到导致TC-NER启动的修复模式？第二，中国的经济状况如何 DNA损伤-受阻的POL II远离DNA损伤，以允许在TC- 内尔入会？第三，是否还有缺失的TC-NER因素有待发现？如果是这样，他们是如何 符合这个几十年来的谜题吗？这项提案的目标是解决以下关键的机械性问题 TC-NER启动。我们建议通过一种创新的混合方法来解决这些具有挑战性的问题 结合了X射线结晶学、低温电子显微镜、计算生物学、生物化学、遗传学和基因组学 方法：研究方法。我们假设CSB在重构病变阻止的POL II和坐标中起重要作用 延长因子和Pol II与其他修复因子的置换促进下游病变 TC-NER启动期间的验证步骤。为了检验这一假设，我们建议研究 病变抑制的POL II复合体、Rad26/CSB和其他转录/修复因子之间的功能相互作用。 我们建议阐明TC-NER启动的神秘机制的分子基础。我们希望 确定参与TC-NER启动的关键蛋白质复合体。我们的项目有三个具体目标： 目的1：确定Rad26/CSB、Spt4/5与DNA损伤相互作用的分子基础。 逮捕了波尔二世情结。目的2：阐明Elf1在TC-NER启动中的作用。目标3：调查如何 病变受阻的POL II在TC-NER启动过程中被取代。这项拟议的研究具有重要意义 开创性的，因为从这项提议中获得的新知识和结构将具有 对DNA修复领域的变革性影响。归根结底，这些知识将为 开发针对癌症和其他人类疾病的新型TC-NER靶向治疗药物。

Project Summary/Abstract The long-term goal of this project is to understand how DNA lesions are recognized and repaired in the actively transcribed genome. Harmful DNA lesions, caused by endogenous and environmental agents, must be promptly recognized and repaired in order to avoid deleterious threats to genome integrity. Transcription- coupled nucleotide excision repair (TC-NER) is an important DNA repair pathway as it removes DNA lesions within the transcribed genome. However, little is known about the molecular mechanism of eukaryotic TC-NER initiation. Cockayne Syndrome B protein (CSB), a master TC-NER coordinator, is recruited to the DNA lesion- arrested Pol II site and plays a key role in the initiation of eukaryotic TC-NER. Previously, we reported the first yeast Pol II-Rad26/CSB ternary complex structure, shedding new lights on this important process. However, there is still a fundamental knowledge gap in understanding what happens after CSB recruitment to the DNA lesion-arrested Pol II. Several long-standing questions in the field remain unanswered. First, how does CSB use its DNA translocase activity to remodel the DNA lesion-arrested Pol II and switch Pol II from the transcription elongation mode to the repair mode that leads to the initiation of TC-NER? Second, how is the DNA lesion-arrested Pol II moved away from the DNA lesion to allow the access of repair proteins during TC- NER initiation? Third, are there any missing TC-NER factors that remain to be discovered? If so, how do they fit into this decades-old puzzle? The objective of this proposal is to address these key mechanistic questions in TC-NER initiation. We propose to tackle these challenging questions with an innovative hybrid approach that combines X-ray crystallography, Cryo-EM, computational biology, biochemistry, genetic, and genomic methods. We hypothesize that CSB plays important roles in remodeling lesion-arrested Pol II and coordinates the displacement of elongation factors and Pol II with other repair factors to promote downstream lesion verification steps during the initiation of TC-NER. To test this hypothesis, we propose to investigate the functional interplays between lesion-arrested Pol II complex, Rad26/CSB, and other transcription/repair factors. We propose to elucidate the molecular basis of the enigmatic mechanism of TC-NER initiation. We expect to determine key protein complexes involved in the initiation of TC-NER. Our project has three Specific Aims: Aim 1: Determine the molecular basis of the interplay between Rad26/CSB, Spt4/5, and the DNA lesion- arrested Pol II complex. Aim 2: Elucidate the role of Elf1 in the initiation of TC-NER. Aim 3: Investigate how the lesion-arrested Pol II is displaced during TC-NER initiation. The proposed research is significant and groundbreaking because novel knowledge and structures obtained from this proposal will have a transformative impact on the field of DNA repair field. Ultimately, such knowledge will provide a framework for developing novel TC-NER targeting therapeutics against cancer and other human diseases.