Biochemical reconstitution and inhibition of TMEJ

TMEJ 的生化重建和抑制

• 批准号: 10468630
• 负责人: RICHARD D WOOD
• 金额: $ 38.24万
• 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/10468630
• 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; 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 该项目将研究哺乳动物DNA聚合酶θ（polθ），定义酶修复DNA 通过聚合酶theta介导的末端连接（TMEJ）的双链断裂。这是项目2（“生化” 对TMEJ的重构和抑制），这是一个计划项目的一部分，标题为“聚合酶Theta，基因组 不稳定性和癌症”。尽管TMEJ具有生物学重要性，但我们对此一无所知 分子机制以及过程中肿瘤中特定漏洞的缺陷。 polθ是一个大的 蛋白质（哺乳动物细胞中的290 kDa）具有DNA聚合酶结构域的独特排列，一个解旋酶 - 像域和连接的中央域。 该项目旨在填补知识的几个主要空白：首先，我们将重点关注Polθ中的功能域 分析DNA微学选择和终端修剪的机制。第二，我们将定义核心 可以执行TMEJ反应的组件。第三，我们将确定如何 癌症影响对POLθ抑制，PARP抑制和DNA损伤敏感性的反应。 在AIM 1“ TMEJ中Polθ的结构活性分析”中，我们将确定允许和最佳的 结束连接的条件，以及独特的polθRestidues和插入环的功能作用，以及候选人 核酸酶用于末端。 在AIM 2中，“重组反应中TMEJ组件的功能”，我们​​将研究最多 PARP和RPA的生物学相关DNA连接酶角色，我们将评估Polθ执行的能力 DNA尾巴与受损坏的碱基的连接过程中的跨质量合成。 在“针对TMEJ途径”的AIM 3中，我们将研究引起Polθ脆弱性的遗传因素 有缺陷的细胞，PARP抑制剂的影响，我们将开始探索polθ抑制剂。新的polθ小 分子抑制剂（2-3个项目的合作）将作为研究工具进行研究。 该研究工作将在计划项目中与其他三个项目进行高度协调 和三个核心。我们联合的潜水方法包括分子生物学，生物化学，结构 生物学和生物物理学。底物，蛋白质和实验将通过项目1、3和4进行设计，并且 通过核心A的反馈不断监控A。 由CoreC的构造C