Biochemical reconstitution and inhibition of TMEJ

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2(“生化” TMEJ的重组和抑制“)，这是名为”聚合酶theta，基因组“计划的一部分 不稳定和癌症“。尽管TMEJ具有重要的生物学意义，但令人惊讶的是，我们对其知之甚少 分子机制以及这一过程中的缺陷如何赋予肿瘤特定的脆弱性。POLθ是一家大型 蛋白质(哺乳动物细胞中为290 kDa)，具有DNA聚合酶结构域的独特排列，解旋酶- 像域，和连接的中心域。 本项目旨在填补知识中的几个主要空白：首先，我们将重点介绍POLθ中的功能域 分析DNA微同源选择和末端修剪的机制。第二，我们将定义核心 可以进行TMEJ反应的组件。第三，我们将确定人类的遗传背景 癌症影响对POLθ抑制、PARP抑制和DNA损伤敏感性的反应。 在目标1《TMEJ中Polθ的结构-活性分析》中，我们将确定允许的和最优的 末端连接的条件，唯一的POLθ残基和插入环的功能作用，以及候选 用于末端修剪的核酸酶。 在目标2“TMEJ组分在重组反应中的作用”中，我们将研究最多 生物学上相关的DNA连接酶在PARP和RPA中的作用，我们将评估POLθ执行 DNA尾部与受损碱基连接过程中的跨损伤合成。 在目标3“以TMEJ途径为靶点”中，我们将调查导致POLθ易损性的遗传因素 有缺陷的细胞，PARP抑制剂的影响，我们将开始探索POLθ抑制剂。全新POLθ小型机 分子抑制剂，一个项目2-3的合作，将被作为研究工具进行调查。 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、蛋白质和实验将通过项目1、3和4以及 通过Core A持续监测反馈。蛋白质纯化将由Core B和细胞系支持 由Core C构建。

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.