Molecular Mechanisms for DNA Damage Processing by Transcription Machinery

项目概要/摘要 该项目的长期目标是了解DNA损伤是如何在细胞中识别和修复的。 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， 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）是一种重要的DNA修复途径，它可以清除DNA损伤 在转录的基因组中。然而，对于真核生物TC-NER的分子机制知之甚少 入会仪式Cockayne综合征B蛋白（CS B），一个主TC-NER协调者，被招募到DNA损伤中- 在真核生物TC-NER的启动中起关键作用。此前，我们报道了第一个 酵母Pol II-Rad 26/CSB三元复合物结构，为这一重要过程提供了新的思路。然而，在这方面， 在理解CSB招募到DNA后会发生什么方面，仍然存在基本的知识差距 病变停滞Pol II.该领域的几个长期问题仍未得到解答。首先，公务员事务局如何 利用其DNA移位酶活性重塑DNA损伤停滞的Pol II，并将Pol II从 转录延伸模式的修复模式，导致启动的TC-NER？第二， DNA损伤阻滞的Pol II从DNA损伤处移开，以允许修复蛋白在TC期间进入。 NER入会仪式？第三，是否有任何缺失的TC-NER因素有待发现？如果是这样， 能解开这个几十年的谜团吗本提案的目的是解决这些关键的机械问题， TC-NER启动。我们建议用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射线晶体学、冷冻电镜、计算生物学、生物化学、遗传学和基因组学 方法.我们推测CSB在重构病变阻滞的Pol II和协调 延伸因子和Pol II与其它修复因子置换以促进下游损伤 TC-NER启动期间的验证步骤。为了验证这一假设，我们建议调查 损伤停滞Pol II复合物、Rad 26/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引发的神秘机制的分子基础。我们期望 确定参与TC-NER启动的关键蛋白质复合物。我们的项目有三个具体目标： 目的1：确定Rad 26/CSB，Spt 4/5和DNA损伤之间相互作用的分子基础。 被逮捕的Pol II复合体目的2：阐明Elf 1在TC-NER启动中的作用。目标3：调查如何 在TC-NER启动期间，病变停滞的Pol II被移位。这项研究意义重大， 开创性的，因为新的知识和结构从这一建议将有一个 对DNA修复领域的变革性影响。最终，这些知识将提供一个框架， 开发针对癌症和其他人类疾病的新型TC-NER靶向治疗剂。

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.