Cellular requirements for Pol theta function

Pol theta 功能的细胞要求

• 批准号: 10640885
• 负责人: DALE A RAMSDEN
• 金额: $ 24.58万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 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/10640885
• 关键词: Acute; 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; Methods; Molecular; Molecular Biology; Monitor; Mutate; Names; Nonhomologous DNA End Joining; Normal Cell; Nucleotides; Pathway interactions; Play; Poison; Polymerase; Rationalization; Research; Role; SET gene; TOP1 gene; Testing; Variant; Work; brca gene; cancer therapy; candidate validation; 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; superresolution microscopy; targeted cancer therapy; whole genome

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 聚合酶 theta 介导的末端连接 (TMEJ) 导致双链断裂。这是项目 1（“蜂窝 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 的机制和生物学作用尚不清楚。重要的是，正常情况下可以容忍 Pol θ 的损失。 细胞，但在许多癌症中是致命的，使其成为癌症治疗的有吸引力的目标。不幸的是，主要 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 用于识别遗传相互作用的强大平台。我们还将开发新方法来探测 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： 目标 1“我们将确定蜂窝 TMEJ 的要求”。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”。我们将评估阻止的程度 另一条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 研究 Pol θ 丢失对化疗药物引起的基因组疤痕的影响 (2B)。 目标 3“我们将采用基因筛选来鉴定 TMEJ 的新介质”。我们将鉴定一组基因 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 三个核心。我们综合了多种方法，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 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。