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.

项目总结