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 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 迅速修复，以避免对基因组完整性的有害威胁。转录偶联DNA修复 (TCR)是一条重要的DNA修复途径，因为它消除了转录基因组中的DNA损伤 转录偶联方式。然而，真核细胞TCR的分子机制仍不清楚。的确有 有证据表明Cockayne综合征B蛋白(CSB)，一个主要的TCR协调者，被招募到DNA损伤- 在真核细胞TCR的启动过程中起着关键作用。然而，存在一个根本性的差距。 了解CSB招募到DNA损伤受阻POL的分子基础II.一个长期存在的 现场的问题是，CSB如何识别并与DNA损伤被阻止的POL II和随后的DNA损伤相互作用 启动TCR。这项建议的目的是阐明CSB在TCR启动中的作用。中环 假设CSB识别特定POL II结构域和核酸的结构特征 在受阻的POL II复合体中的支架，经历了显著的构象变化，并受到 通过其自身的监管主题进行自动监管。这一假设是在初步的基础上提出的 在申请人的实验室中产生的数据，将通过执行结构和功能测试 CSB与被阻挡的POL II络合物相互作用的表征。这种方法是 创新，因为它利用了一种新的混合方法，结合了X射线结晶学，冷冻-EM， 生物物理学、生物化学和遗传学。这项拟议的研究具有重大意义和开创性，因为 从这项提议中获得的新知识和新结构将对 DNA修复和垂直推进对转录偶联修复是如何启动的理解。最终， 这些知识将为开发新型TCR靶向治疗癌症和 其他人类疾病。

Project Summary/Abstract The long-term goal of this project is to understand how DNA lesions are recognized and repaired in the transcribed genome. Harmful DNA lesions, caused by endogenous and environmental agents, must be promptly repaired in order to avoid deleterious threats to genome integrity. Transcription-coupled DNA repair (TCR) is an important DNA repair pathway because it removes DNA lesions within the transcribed genome in a transcription-coupled manner. However, the molecular mechanism of eukaryotic TCR remains elusive. There is evidence that Cockayne Syndrome B protein (CSB), a master TCR coordinator, is recruited to the DNA lesion- arrested Pol II site and plays a key role in the initiation of eukaryotic TCR. However, there is a fundamental gap in understanding the molecular basis of CSB recruitment to the DNA lesion-arrested Pol II. A long-standing question in the field is how CSB recognizes and interacts with the DNA lesion-arrested Pol II and subsequently initiates TCR. The objective of this proposal is to elucidate the roles of CSB in TCR initiation. The central hypothesis is that CSB recognizes the structural features of specific Pol II domains and the nucleic acid scaffold in arrested Pol II complexes, undergoes significant conformational changes, and is subject to autoregulation by its own regulatory motifs. This hypothesis has been formulated on the basis of preliminary data produced in the applicants' laboratories and will be tested by performing structural and functional characterizations of the interactions between CSB and the arrested Pol II complex. This approach is innovative, because it utilizes a novel hybrid method that combines X-ray crystallography, Cryo-EM, biophysics, biochemistry, and genetics. 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 and vertically advance the understanding of how transcription-coupled repair is initiated. Ultimately, such knowledge will provide a framework for developing novel TCR targeting therapeutics against cancer and other human diseases.