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; 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.

项目摘要 该项目将研究哺乳动物DNA聚合酶θ（polθ），定义酶修复DNA 通过聚合酶theta介导的末端连接（TMEJ）的双链断裂。这是项目1（“蜂窝” polθ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，polθ的丢失在正常状态下被允许 细胞但在许多癌症中都是致命的，使其成为癌症治疗的吸引力。不幸的是，少校 在作用机理和POLθ的生物学作用机理中的知识差距都削弱了发展 安全，有效的目标策略。 项目1将通过利用现有的专业蜂窝测定和A来确定POLθ功能的要求 可靠的平台来识别遗传相互作用。我们还将开发新方法来探测 Polθ在细胞修复中的不同作用。 该项目旨在填补知识的几个主要空白： 在AIM 1中，“我们将确定对蜂窝TMEJ的要求”。我们将在特定的POL中使用细胞系有缺陷 Q活动和与TMEJ相关的其他因素，并评估这些缺陷对细胞TMEJ的影响。 在AIM 2中，“我们将确定吸引TMEJ的细胞环境”。我们将评估哪些障碍的程度 另一个维修途径的最新步骤使TMEJ（2A）参与。我们还将使用高吞吐量测序 研究Polθ损失对化学治疗剂引起的基因组疤痕的影响（2B）。 在AIM 3中“我们将采用遗传屏幕来识别TMEJ的新介体”。我们将确定一组基因 通过找到那些在突变时在一系列细胞中致命的基因，在一系列有缺陷的细胞中，该基因是相同的途径 其他修复途径使它们急性依赖于TMEJ的生存。 该研究工作将在计划项目中与其他三个项目以及 三个核心。我们联合的潜水方法包括分子生物学，生物化学，结构生物学和 生物物理学。我们将采用突变体并根据与项目2、3和4共享的结果启动实验。 通过核心A的反馈将不断监控。蛋白质纯化将由核心B支持 核心C的细胞系结构。