Cellular requirements for Pol theta function

Pol theta 功能的细胞要求

基本信息

批准号：
10468629
负责人：
DALE A RAMSDEN
金额：
$ 25.03万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
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/10468629
关键词：
Acute Address Biochemical Biochemistry Biological Biological Assay Biophysics CRISPR screen Camptothecin Cell Line Cell Survival Cells Cellular Assay Chromosomal Breaks Cicatrix DNA Damage DNA Double Strand Break DNA Repair Enzymes DNA Repair Gene DNA replication fork DNA-Directed DNA Polymerase Defect Development Enzymes Feedback G22P1 gene Genes Genetic Genetic Epistasis Genetic Screening Genome Genomic Instability Genomics High-Throughput Nucleotide Sequencing In Vitro Inherited Knowledge Link Malignant Neoplasms Mediating Mediator of activation protein Methods Microscopy Molecular Molecular Biology Monitor Mutate Names Nonhomologous DNA End Joining Normal Cell Nucleotides Pathway interactions Play Poison Polymerase Proteins Research Resolution Role SET gene TOP1 gene Testing Variant Work base brca gene cancer therapy cell growth chemotherapeutic agent experimental study extrachromosomal DNA genome-wide homologous recombination interest live cell imaging member multidisciplinary mutant novel p53-binding protein 1 programs protein purification reconstitution repaired response single molecule structural biology targeted cancer therapy vif Genes whole genome

项目摘要

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.

项目概要该项目将研究哺乳动物 DNA 聚合酶 θ (Pol θ)，它是 DNA 修复的定义酶通过聚合酶 theta 介导的末端连接 (TMEJ) 实现双链断裂这是项目 1（“细胞”）。 Pol θ 的要求”），这是一个名为“聚合酶 theta、基因组不稳定性和癌症”。 TMEJ 机制和生物学作用尚不清楚，重要的是，正常人可以容忍 Pol θ 的丢失。细胞，但在许多癌症中是致命的，这使其成为癌症治疗的一个有吸引力的目标。 Pol θ 的作用机制和生物学作用方面的知识差距削弱了其发展安全、有效的目标策略。项目 1 将通过利用现有的专门细胞测定和我们还将开发新的方法来探测遗传相互作用。 Pol θ 在细胞修复中的不同作用。该项目旨在填补几个主要的知识空白：在目标 1“我们将确定细胞 TMEJ 的要求”中，我们将使用特定 Pol 有缺陷的细胞系。 q 与 TMEJ 相关的活动和其他因素，并评估这些缺陷对细胞 TMEJ 的影响。在目标 2 中，“我们将确定参与 TMEJ 的细胞环境”，我们将评估阻碍的程度。另一种修复途径的后期步骤涉及 TMEJ (2A)，我们还将采用高通量测序来进行修复。研究 Pol θ 丢失对化疗药物引起的基因组疤痕的影响 (2B)。在目标 3 中，“我们将采用基因筛选来鉴定 TMEJ 的新介质”。与 Pol θ 相同的途径，通过找到那些突变时在一组有缺陷的细胞中致命的基因其他修复途径使它们严重依赖 TMEJ 生存。研究工作将在该计划项目内与其他三个项目和我们的综合多种方法包括分子生物学、生物化学、结构生物学和我们将使用突变体并根据与项目 2、3 和 4 共享的结果启动实验，这将通过核心 A 的反馈进行持续监控。蛋白质纯化将由核心 B 支持，以及 Core C 构建的细胞系。