Cellular requirements for Pol theta function

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1(“细胞” 对POLθ的要求“)，这是一个项目的一部分，名为”聚合酶theta，基因组不稳定性和 癌症“。 TMEJ的机制和生物学作用尚不清楚。重要的是，POLθ的丢失在正常情况下是可以容忍的 但在许多癌症中是致命的，使其成为癌症治疗的一个有吸引力的靶点。不幸的是，少校 POLθ的作用机制和生物学作用方面的知识差距削弱了这一发展 安全、有效的目标策略。 项目1将通过利用现有的专业细胞分析和 用于识别遗传交互作用的强大平台。我们还将开发新的方法来探索 POLθ在细胞修复中的不同作用。 该项目旨在填补知识方面的几个主要空白： 在目标1“我们将确定蜂窝TMEJ的要求”。我们将在特定的极点使用有缺陷的细胞系 Q活动和其他与TMEJ相关的因素，并评估这些缺陷对细胞TMEJ的影响。 在目标2“我们将确定参与TMEJ的蜂窝环境”。我们将评估阻塞的程度 另一条修复途径的后期步骤与TMEJ(2A)接合。我们还将采用高通量测序来 研究POLθ缺失对化疗药物(2B)引起的基因组疤痕的影响。 在目标3中，“我们将利用基因筛查来确定TMEJ的新介体”。我们将确定一组基因 与Polθ相同的途径是，通过在一组有缺陷的细胞中发现那些当突变时是致命的基因 其他修复途径使他们严重依赖TMEJ生存。 研究工作将在计划项目内与其他三个项目和 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。我们将使用突变体，并根据与项目2、3和4分享的结果启动实验， 它将通过核心A的反馈进行持续监测，蛋白质纯化将由核心B支持， 和CoreC构建细胞系。

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 1 (“Cellular requirements for Pol θ”) which is part of a Program Project titled, “Polymerase theta, genome instability, and cancer”. TMEJ mechanism and biological role is not well understood. Importantly, loss of Pol θ is tolerated in normal cells but is lethal in many cancers, making it an attractive target for cancer therapy. Unfortunately, the major knowledge gaps in both the mechanism of action and biological role for Pol θ have undercut the development of safe, effective targeting strategies. Project 1 will identify requirements for Pol θ function by leveraging existing specialized cellular assays and a robust platform for identifying genetic interactions. We will additionally develop new methods to probe the different roles of Pol θ in cellular repair. This project aims to fill several major gaps in knowledge: In Aim 1 “We will identify requirements for cellular TMEJ”. We will use cell lines defective in specific Pol q activities and other factors linked to TMEJ, and assess the impact of these defects on cellular TMEJ. In Aim 2 “We will determine cellular contexts that engage TMEJ”. We will assess the extent to which blocks to late steps in another repair pathway engage TMEJ (2A). We will also employ high throughput sequencing to investigate the impact of Pol θ loss on genomic scars caused by a chemotherapeutic agent (2B). In Aim 3 “We will employ a genetic screen to identify new mediators of TMEJ”. We will identify a set of genes in the same pathway as Pol θ, by finding those genes that when mutated are lethal in a panel of cells defective in other repair pathways that renders them acutely dependent on TMEJ for survival. 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. We will employ mutants and initiate experiments based on results shared with projects 2, 3, and 4, which will be constantly monitored with feedback via Core A. Protein purification will be supported by Core B, and cell line construction by Core C.