Single-molecule studies of Theta mediated end joining

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4（“单分子 Studies of TMEJ”），其是标题为“Polymerase theta，genome instability，and cancer”的计划项目的一部分。 尽管TMEJ在生物学上很重要，但我们对其分子机制以及如何作用知之甚少。 该过程中的缺陷赋予肿瘤特定的脆弱性。Pol θ是一种大分子蛋白质（在哺乳动物中为290 kDa）， 细胞）具有DNA聚合酶结构域、解旋酶样结构域和连接结构域的独特排列。 中央域。 这个项目旨在填补我们对TMEJ的认识中的几个基本空白，并探索新的假设 通过采用一系列创新的生物化学、细胞和单分子技术和测定， 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，以及它如何被其他修复因子调节。 第二，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如何帮助修复折叠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“通过生化重建系统的TMEJ突触机制”中，我们将建立 Pol θ解旋酶、聚合酶和其他结构域对初始链配对的特定贡献 活性、微同源性搜索以及与NHEJ和HR的串扰 在目标2，“Interplay of TMEJ with NHEJ at DSB sites in cells”中，我们将测量TMEJ与NHEJ在细胞中DSB位点的相互作用的特定模式。 作为DDR和规范DSB功能的TMEJ修复中间体的招募-排除和组织 修复，以及它们如何受到关键修复缺陷的影响。 在目标3中，“TMEJ在修复折叠的复制叉中的作用”，我们将研究Pol蛋白在修复折叠的复制叉中的作用。 单端DSB（seDSB）在折叠的复制叉和有毒次级的分解中形成 结构. 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1，2和3， 将通过核心A不断监测反馈。蛋白纯化将由核心B和细胞支持 核心C的线路建设。

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.