Transcription-Coupled & Replication-Associated Excision Repair

转录偶联

• 批准号: 7152382
• 负责人: Priscilla K. Cooper
• 金额: $ 6.27万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7152382
• 关键词: DNA; neoplasm /cancer; proteins

The Project (Transcription-Coupled and Replication-Associated Excision Repair) focuses on mechanisms coupling DNA excision repair machinery with transcription and replication. Both Nucleotide Excision Repair (NER) and Base Excision Repair (BER) are highly coordinated by interactions between proteins in the pathway. Moreover, they are preferentially targeted by specialized transcriptioncoupled repair (TCR) machinery to lesions that affect transcription elongation or by replication-associated repair (RAR) to lesions near the replication fork or in recently replicated DNA. We hypothesize that these interactions and their effects on function are regulated through unstructured flexible regions that undergo disorder-to-order transformations upon complex formation and/or post-translational modifications. We will test this overall hypothesis and specific hypotheses in five Aims by collaborative studies to characterize, validate, and map interactions, identify damage-induced modifications, observe effects of complexes on DNA structure by scanning force microscopy (SFM), and visualize subunits and complexes by electron microscopy (EM), small angle X-ray scattering (SAXS), and protein crystallography (PX). Aim 1 will structurally characterize early steps of TCR: recognition by XPG and CSB of RNA Polymerase II (RNAPII) stalled at a lesion, and remodeling of RNAPII by TFIIH to allow access to the lesion. SFM and EM studies will test the hypothesis that these occur by ordered conformational changes. Aim 2 will structurally characterize CSB and reinvestigate its causal role in CS by determining whether mutant CSB interferes with responses to oxidative DNA damage through non-productive interactions with other proteins in the pathway. Aim 3 will investigate the identified interactions that couple BER and NER to transcription through (a) SAXS and PX studies of XPG protein and its domains and complexes, (b) analysis of interactions of NEIL2 with RNAPII, XPG and CSB, and (c) characterization of the effect of post-translational modifications on XPG and NEIL2 interactions. Aim 4 will characterize the structural basis for BER pathway coordination by interactions of NEIL1 and NEIL2 glycosylases with downstream BER proteins and test the hypothesis that BER pathway progression results in progressive DNA bending. Aim 5 will investigate molecular mechanisms of RAR by determining the structure of the checkpoint sliding clamp -- the 9-1-1 complex - and by characterizing interactions of the MYH and NEIL1 glycosylases with PCNA and 9-1-1. The anticipated outcome is a molecular understanding of cancer predispositions and developmental disorders that arise from defects in the coordination of excision repair with transcription and replication. Collaborations of Project 2 within SBDR and with the UCSF Comprehensive Cancer Center will relate results of these studies to genome integrity and cancer etiology as well as to development of promising molecular targets for cancer drug discovery.

该项目（转录耦合和复制相关切除修复）的重点是耦合DNA切除修复机制与转录和复制。两 核苷酸切除修复（NER）和碱基切除修复（BER）通过相互作用高度协调 蛋白质之间的相互作用此外，它们优先被专门的转录偶联靶向， 修复（TCR）机制的损伤，影响转录延伸或复制相关的 修复（RAR）复制叉附近或最近复制的DNA损伤。我们假设这些 相互作用及其对功能的影响是通过非结构化的柔性区域进行调节的， 复合物形成和/或翻译后修饰时的无序到有序转化。我们将 通过合作研究来测试这一总体假设和五个目标中的具体假设， 验证和绘制相互作用，识别损伤诱导的修饰，观察复合物对DNA的影响 通过扫描力显微镜（SFM）进行结构化，并通过电子显微镜观察亚基和复合物 显微镜（EM）、小角X射线散射（SAXS）和蛋白质晶体学（PX）。目标1将 TCR早期步骤结构表征：RNA聚合酶II（RNAPII）的XPG和CSB识别 在病变处停滞，并通过TFIIH重塑RNAPII以允许进入病变。SFM和EM研究 将测试这些发生有序构象变化的假设。目标2将在结构上 通过确定突变型CSB是否干扰 通过与途径中其他蛋白质的非生产性相互作用对氧化DNA损伤的反应。 目的3将通过（a）SAXS研究BER和NER与转录的相互作用 （B）分析NEIL 2与XPG蛋白及其结构域和复合物的相互作用， RNAPII、XPG和CSB的翻译后修饰的作用的表征，以及（c）XPG和CSB的翻译后修饰的作用的表征。 NEIL 2相互作用。目标4将通过相互作用来表征BER途径协调的结构基础 NEIL 1和NEIL 2糖基化酶与下游BER蛋白的关系，并检验BER途径 进展导致进行性DNA弯曲。目的5将探讨RAR的分子机制， 确定检查点滑动夹的结构-9-1-1复合物-并通过表征 MYH和NEIL 1糖基化酶与PCNA和9-1-1.预期的结果是 对癌症易感性和发育障碍的分子理解， 切除修复与转录和复制的协调。SBDR内项目2的合作 并与加州大学旧金山分校综合癌症中心将这些研究的结果与基因组的完整性， 癌症病因学以及用于癌症药物发现的有前景的分子靶点的开发。