Transcription-Coupled & Replication-Associated Excision Repair

转录偶联

• 批准号: 7924234
• 负责人: Priscilla K. Cooper
• 金额: $ 35.54万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7924234
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affect; Antineoplastic Agents; Apoptosis; Atomic Force Microscopy; Base Excision Repairs; Binding; Cancer Etiology; Catalytic Domain; Cells; Cellular biology; Cockayne Syndrome; Collaborations; Complex; Comprehensive Cancer Center; Coupled; Coupling; Crystallography; DNA; DNA Damage; DNA Repair; DNA Structure; Defect; Development; Disease; Electron Microscopy; Excision Repair; Figs - dietary; Genetic Transcription; Genome; Health; Human; Imagery; Lead; Lesion; Ligase; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Modification; Molecular; Molecular Target; Mutagenesis; Mutation; N-terminal; Nucleotide Excision Repair; Nucleotides; Outcome; Pain; Pathway interactions; Patients; Phosphorylation; Post-Translational Protein Processing; Predisposition; Process; Proteins; RNA Polymerase II; Recombinants; Roentgen Rays; Role; Rotation; SWI2/SNF2; Signal Transduction; Slide; Structure; Testing; Transcription Elongation; Transcription-Coupled Repair; UV sensitive syndrome; XPGC protein; XRCC1 gene; base; deprivation; developmental disease; drug discovery; flexibility; ilium; mutY adenine glycosylase; mutant; oxidative DNA damage; oxidative damage; protein complex; protein protein interaction; repair enzyme; repaired; response; stoichiometry; transcription factor TFIIH; ultraviolet damage

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的切除修复）着重于将DNA切除修复机械与转录和复制耦合的机制。两个都 核苷酸切除修复（NER）和碱基切除修复（BER）高度协调 在路径中的蛋白质之间。此外，它们优先通过专门的转录耦合来靶向 修复（TCR）机械到影响转录伸长或通过复制相关的病变 修复（RAR）对复制叉附近或最近重复的DNA附近的病变。我们假设这些 相互作用及其对功能的影响通过非结构化的灵活区域进行调节 复杂形成和/或翻译后修改后的疾病转化。我们将 通过协作研究表征， 验证和映射相互作用，识别损伤引起的修饰，观察复合物对DNA的影响 通过扫描力显微镜（SFM）结构，并通过电子可视化亚基和复合物 显微镜（EM），小角度X射线散射（SAXS）和蛋白质晶体学（PX）。目标1意志 结构表征了TCR的早期步骤：XPG识别RNA聚合酶II（RNAPII）的CSB识别 停滞在病变上，然后通过TFIIH对RNAPII进行改建，以允许进入病变。 SFM和EM研究 将检验以下假设：这些假设是通过有序构象变化发生的。 AIM 2将在结构上 表征CSB并通过确定突变体CSB是否干扰CS中的CS中的因果作用 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。 AIM 3将调查通过（a）saxs将Ber和Ner与转录的确定相互作用 XPG蛋白及其结构域和复合物的PX研究，（b）Neil2与 RNAPII，XPG和CSB，以及（c）翻译后修饰对XPG和 Neil2相互作用。 AIM 4将通过相互作用来表征BER途径协调的结构基础 Neil1和Neil2糖基酶与下游BER蛋白，并测试BER途径的假设 进展会导致进行性DNA弯曲。 AIM 5将研究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NEIL1糖基酶与PCNA和9-1-1的相互作用。预期的结果是 对癌症易感和发育障碍的分子理解是由缺陷引起的 与转录和复制的切除修复协调。 SBDR中项目2的合作 随着UCSF综合癌症中心将将这些研究的结果与基因组完整性联系起来 癌症病因以及开发有希望的分子靶标的癌症药物发现。