Transcription-Coupled & Replication-Associated Excision Repair

转录偶联

基本信息

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
项目类别：
财政年份：
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

项目摘要

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（转录偶联和复制相关切除修复）重点研究 DNA 切除修复机制与转录和复制的耦合机制。两个都核苷酸切除修复（NER）和碱基切除修复（BER）通过相互作用高度协调途径中的蛋白质之间。此外，它们优先被专门的转录偶联靶向修复（TCR）机制对影响转录延伸或复制相关损伤的损伤对复制叉附近或最近复制的 DNA 中的损伤进行修复 (RAR)。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灵活区域来调节的，这些区域经历复杂形成和/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检验这一总体假设和五个目标中的具体假设，以表征，验证和绘制相互作用图谱，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) 观察结构，并通过电子可视化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将结构表征 TCR 的早期步骤：XPG 和 CSB 对 RNA 聚合酶 II (RNAPII) 的识别停滞在病变处，并通过 TFIIH 重塑 RNAPII 以允许到达病变处。 SFM 和 EM 研究将检验这些是通过有序构象变化发生的假设。目标 2 将在结构上通过确定突变 CSB 是否干扰来表征 CSB 并重新研究其在 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来应对氧化 DNA 损伤。目标 3 将研究已确定的通过 (a) SAXS 将 BER 和 NER 与转录耦合的相互作用 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 CSB 影响的表征 NEIL2 相互作用。目标 4 将通过相互作用来表征 BER 通路协调的结构基础 NEIL1 和 NEIL2 糖基化酶与下游 BER 蛋白的结合，并测试 BER 通路的假设进展导致DNA逐渐弯曲。目标 5 将通过以下方式研究 RAR 的分子机制：确定检查点滑动夹具的结构——9-1-1复合体——并通过表征 MYH 和 NEIL1 糖基化酶与 PCNA 和 9-1-1 的相互作用。预期结果是对癌症易感性和由缺陷引起的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合作，将这些研究结果与基因组完整性和癌症病因学以及开发用于癌症药物发现的有前景的分子靶标。