Single-molecule studies of Theta mediated end joining

Theta 介导的末端连接的单分子研究

• 批准号: 10468632
• 负责人: Eli Rothenberg
• 金额: $ 38.15万
• 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/10468632
• 关键词: Address; Affect; Biochemical; Biochemistry; Biological; Biological Assay; Biophysics; Camptothecin; Cell Line; Cells; Cellular Assay; Chromosomal Breaks; Chromosome Pairing; Collaborations; Complex; Conflict (Psychology); DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Enzymes; DNA Structure; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Data; Defect; Exclusion; Experimental Designs; Feedback; Genomic Instability; Human; Human Cell Line; In Vitro; Individual; Kinetics; Knowledge; Length; Malignant Neoplasms; Mammalian Cell; Measurement; Measures; Mediating; Microscopy; Molecular; Molecular Biology; Monitor; Mutation; Nonhomologous DNA End Joining; Pathway interactions; Patients; Polymerase; Process; Property; Proteins; Reaction; Recombinant Proteins; Regulation; Research; Resolution; Role; Site; Structure; System; Techniques; Testing; TimeLine; Variant; Visualization; Wood material; Work; cancer cell; design; experimental study; helicase; innovation; mutant; novel; p53-binding protein 1; programs; protein function; protein purification; reconstitution; recruit; repaired; single molecule; structural biology; therapy resistant; tool; treatment response; 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 4 (“Single-molecule studies 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 fundamental gaps in our knowledge of TMEJ, and explore novel hypotheses by employing an array of innovative biochemical, cellular, and single-molecule techniques and assays to define the key steps and molecular mechanisms of TMEJ. First, we will focus on the initial elusive steps of TMEJ including synapsis and DNA microhomology search process, and how it is modulated by other repair factors. Second, we will define the kinetics and regulation of TMEJ during cellular DSB repair. Third, we will establish how TMEJ contributes to repair of collapsed replication forks and repair of replication conflicts at secondary DNA structures. In Aim 1 “Mechanism of TMEJ synapsis via biochemically reconstituted system” we will establish the specific contributions of Pol θ helicase, polymerase and other structural domains for initial strand pairing activity, micro-homology search, and crosstalk with NHEJ and HR In Aim 2, “Interplay of TMEJ with NHEJ at DSB sites in cells”, we will measure the specific modes of recruitment-exclusion and organization of TMEJ repair intermediates as a function of DDR and canonical DSB repair, and how they are affected by key repair deficiencies. In Aim 3, “TMEJ role(s) in repair of collapsed replication forks”, we will investigate the roles of Pol  in repair of single-ended DSBs (seDSB) formed at collapsed replication forks and resoltuon of toxic secondary structures. 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, 2, and 3, and will be constantly monitored with feedback via Core A. Protein purification will be supported by Core B, and cell line construction by Core C.

项目总结 这个项目将研究哺乳动物DNA聚合酶θ(POLθ)，这是DNA修复的决定酶 聚合酶Theta介导的末端连接(TMEJ)导致双链断裂。这是项目4(“单分子 TMEJ的研究“)，这是一个项目的一部分，名为”聚合酶theta，基因组不稳定性，与癌症“。 尽管TMEJ具有重要的生物学意义，但令人惊讶的是，我们对其分子机制和如何 这一过程中的缺陷赋予了肿瘤特定的脆弱性。POLθ是哺乳动物体内的一种大蛋白(290kDa) 细胞)具有独特的DNA聚合酶结构域、解旋酶样结构域和连接 中心域。 这个项目旨在填补我们对TMEJ知识的几个基本空白，并探索新的假设 通过采用一系列创新的生化、细胞和单分子技术和分析来确定 TMEJ的关键步骤和分子机制。首先，我们将关注TMEJ最初难以捉摸的步骤 包括突触和DNA微同源搜索过程，以及它是如何被其他修复因子调制的。 其次，我们将定义TMEJ在细胞DSB修复过程中的动力学和调节。第三，我们将建立 TMEJ如何帮助修复崩溃的复制分叉和修复辅助服务器上的复制冲突 DNA结构。 在目标1“TMEJ通过生化重组系统的突触机制”中，我们将建立 POLθ解旋酶、聚合酶和其他结构域对初始链配对的特异性贡献 NHEJ和HR的活性、微观同源搜索和串扰 在目标2“TMEJ与NHEJ在细胞中DSB位点的相互作用”中，我们将测量 招募-排除和组织TMEJ修复中间体作为DDR和规范DSB的功能 修复，以及它们如何受到关键修复缺陷的影响。 在目标3中，我们将研究POL在修复倒塌的复制叉子中的作用 在折叠复制叉处形成的单端DSB(SeDSB)和有毒次生物质的解决 结构。 研究工作将在方案项目内与其他三个项目高度协调 和三个核心。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。底物、蛋白质和实验将与项目1、2和3一起设计，以及 将通过核心A持续监测反馈。蛋白质纯化将由核心B和细胞支持 用C芯进行线路建设。