Polymerase Theta Mediated End Joining: Mechanism and Essential Functions in Repair of Chromosome Breaks

项目摘要/摘要 染色体断裂的修复对正常细胞生长、预防和促进癌症至关重要-- 导致基因组重排，并决定许多癌症治疗的有效性。在最近的工作中 我们的研究小组发现DNA聚合酶theta参与了一条修复染色体断裂的途径，这种途径有利于短 序列重复(微同源)。在目标1中，我们将概述Theta介导的末端连接(TMEJ)的步骤 -其分子机制-并确定每一步的要求。我们将采用以下组合 确定了染色体外底物，分析了靶向染色体断裂的修复，并对 当细胞在不同步骤有缺陷时，对细胞生长和活力的影响的评估。在《目标2》中我们将 阐明Theta介导的末端连接与染色体修复的另一种途径的关系 断裂，同源重组。具体地说，我们将研究BRCA1和53BP1基因的缺陷是如何 参与TMEJ和同源重组共同的早期步骤，影响在 通过这两条路径进行修复。然后，我们还将讨论TMEJ途径作为替代途径的重要性 对于Holliday连接的分辨，是同源重组途径特有的晚期步骤。我们将利用 染色体末端结构和修复的分析，以及通过使用 无脊椎动物模型D黑腹鼠。在目标3中，我们将系统地寻找与 聚合酶theta对一般细胞活力和细胞对电离辐射的抗性都很重要。 我们将使用基于慢病毒载体的CRISPR文库，该文库被策划为专注于310个基因的子集 与DNA损伤反应相关。拟议中的实验将说明一个重要但迄今尚未完成的 鲜为人知的途径工作，特别是何时和为什么它代表的最佳解决方案 在染色体断裂修复过程中遇到的问题。

Project Summary/Abstract Repair of chromosome breaks is essential for normal cell growth, protects against and promotes cancer- causing genome rearrangements, and determines the effectiveness of many cancer therapies. In recent work our group implicated DNA polymerase theta in a pathway for repairing chromosome breaks that favors short sequence repeats (microhomologies). In Aim 1 we will outline the steps in Theta mediated end joining (TMEJ) – its molecular mechanism - and determine the requirements for each step. We will employ a combination of defined extrachromosomal substrates, analysis of repair of a targeted chromosomal break, and quantitative assessments of effects on cell growth and viability when cells are defective in various steps. In aim 2 we will clarify the relationship between Theta mediated end joining and another pathway for repair of chromosome breaks, homologous recombination. Specifically, we will investigate how defects in BRCA1 and 53BP1, genes involved in early steps common to both TMEJ and homologous recombination, impact the choice between repair by these two pathways. We will then also address the importance of the TMEJ pathway as an alternative to Holliday junction resolution, a late step specific to the homologous recombination pathway. We will exploit assays for chromosomal end structure and repair, as well as genetic tools available through use of the invertebrate model D. melanogaster. In aim 3 we will systematically look for novel genetic interactions with Polymerase theta that are important for both general cell viability and cellular resistance to ionizing radiation. We will employ a lentiviral vector based CRISPR library that was curated to focus on a subset of 310 genes relevant to the DNA damage response. The proposed experiments will speak to how an important but as-yet poorly understood pathway works, and especially when and why it represents the best solution to various problems encountered in the course of repair of chromosome breaks.