Biochemical reconstitution and inhibition of TMEJ

TMEJ 的生化重建和抑制

• 批准号: 10202521
• 负责人: RICHARD D WOOD
• 金额: $ 15.73万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10202521
• 关键词: BRCA1 gene; Biochemical; Biochemistry; Biological; Biophysics; Camptothecin; Cell Line; Cells; Cellular Assay; Collaborations; DNA; DNA Damage; DNA Double Strand Break; DNA Insertion Elements; DNA Ligases; DNA Polymerase Inhibitor; DNA Repair Enzymes; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Defect; Enzymes; Excision; Feedback; Future; Genes; Genetic; Genomic Instability; Genomics; High-Throughput DNA Sequencing; Human; Image; Ionizing radiation; Knowledge; Length; Location; Malignant Neoplasms; Mammalian Cell; Mediating; Modeling; Molecular; Molecular Biology; Monitor; Mutate; Mutation; Outcome; Outcome Study; PARP inhibition; Pathway interactions; Polymerase; Process; Proteins; Radiation; Reaction; Research; Resected; Role; SS DNA BP; Structure; Tail; Variant; Work; base; cancer therapy; clinically relevant; design; experimental study; helicase; in vivo Model; inhibitor/antagonist; multidisciplinary; nuclease; pre-clinical; programs; protein purification; reconstitution; repaired; response; small molecule inhibitor; structural biology; synthetic construct; tool; tumor

PROJECT SUMMARY This project will investigate mammalian DNA polymerase θ (Pol θ), the defining enzyme for repair of DNA double-strand breaks by polymerase theta-mediated end joining (TMEJ). This is Project 2 (“Biochemical reconstitution and inhibition of TMEJ”) which is part of a Program Project titled, “Polymerase theta, genome instability, and cancer”. Despite the biological importance of TMEJ, we know surprisingly little about its molecular mechanism and how defects in the process confer specific vulnerabilities in tumors. Pol θ is a large protein (290 kDa in mammalian cells) with a distinctive arrangement of a DNA polymerase domain, a helicase- like domain, and a connecting central domain. This project aims to fill several major gaps in knowledge: First, we will focus on functional domains in Pol θ to analyze the mechanism of DNA microhomology selection and end-trimming. Second, we will define core components that can carry out the TMEJ reaction. Third, we will determine how the genetic background of cancers influences the response to Pol θ suppression, PARP inhibition, and DNA damage sensitivity. In Aim 1 “Structure-activity analysis of Pol θ in TMEJ”, we will determine the permitted and optimal conditions for end joining, and functional roles of unique Pol θ residues and insertion loops, and candidate nucleases for end-trimming. In Aim 2, “Function of TMEJ components in a reconstituted reaction”, we will investigate the most biologically relevant DNA ligases roles for PARP and RPA, and we will assess the ability of Pol θ to perform translesion synthesis during joining of DNA tails with damaged bases. In Aim 3, “Targeting the TMEJ pathway”, we will investigate genetic factors that cause vulnerability in Pol θ defective cells, the influence of PARP inhibitors, and we will begin to explore Pol θ inhibitors. New Pol θ small molecule inhibitors, a Project 2-3 collaboration, will be investigated as research tools. The research work will be highly coordinated within the Program Project with the other three Projects and the three Cores. Our combined diverse approaches include molecular biology, biochemistry, structural biology, and biophysics. Substrates, proteins, and experiments will be designed with Projects 1, 3, and 4 and constantly monitored with feedback via Core A. Protein purification will be supported by Core B, and cell line construction by Core C.

项目摘要 本计画将探讨哺乳动物DNA聚合酶θ（Pol θ），DNA修复的定义酵素 通过聚合酶θ介导的末端连接（TMEJ）的双链断裂。这是项目2（“生物化学 TMEJ的重建和抑制”），其是名为“聚合酶θ，基因组 不稳定和癌症”。尽管TMEJ在生物学上很重要，但我们对其知之甚少， 分子机制以及该过程中的缺陷如何赋予肿瘤特定的脆弱性。Pol θ是一个大的 蛋白质（哺乳动物细胞中290 kDa），具有DNA聚合酶结构域、解旋酶- 类似域和连接中心域。 这个项目旨在填补几个主要的知识空白：首先，我们将关注Pol θ中的功能域 分析DNA微同源选择和末端修剪的机制。其次，我们将定义核心 可以进行TMEJ反应的组分。第三，我们将确定如何遗传背景的 癌症影响对Pol θ抑制、PARP抑制和DNA损伤敏感性的反应。 在目标1“TMEJ中Pol θ的结构-活性分析”中，我们将确定允许的和最佳的 末端连接的条件，以及独特的Pol θ残基和插入环的功能作用，以及候选物 用于末端修剪的核酸酶。 在目标2“重构反应中TMEJ组分的功能”中，我们将研究最重要的 生物学相关的DNA连接酶的PARP和RPA的作用，我们将评估Pol θ的能力， 在DNA尾与受损碱基连接期间的跨损伤合成。 在目标3“靶向TMEJ通路”中，我们将研究导致Pol θ易感性的遗传因素。 缺陷细胞，PARP抑制剂的影响，我们将开始探索Pol θ抑制剂。新Pol θ small 分子抑制剂，项目2-3合作，将作为研究工具进行研究。 研究工作将在计划项目内与其他三个项目高度协调 还有三个核心我们结合了多种方法，包括分子生物学，生物化学，结构 生物学和生物物理学。基质，蛋白质和实验将设计与项目1，3和4， 通过核心A不断监测反馈。蛋白纯化将由核心B和细胞系支持 核心C的构建