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

聚合酶 Theta 介导的末端连接：染色体断裂修复的机制和基本功能

基本信息

批准号：
10170291
负责人：
DALE A RAMSDEN
金额：
$ 46.56万
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：
2018
资助国家：
美国
起止时间：
2018-06-15 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10170291
关键词：
Address Animal Model BRCA1 gene BRCA2 gene Biological Assay Breast Cancer Model CRISPR library Cell Survival Cells Chromosomal Breaks Chromosomes Clinical Clustered Regularly Interspaced Short Palindromic Repeats Cruciform DNA DNA Damage DNA-Directed DNA Polymerase Data Defect Development Embryo Engineering Equilibrium Excision Fibroblasts Genes Genetic Genetic Epistasis Genome Genomic Instability Hereditary Breast and Ovarian Cancer Syndrome Holliday Junction Resolvases Interruption Invertebrates Investigation Ionizing radiation Kinetics Lentivirus Vector Loss of Heterozygosity Malignant Neoplasms Mediating Mitotic Modeling Molecular Mus Mutation Names Nonhomologous DNA End Joining Normal Cell Pathway interactions Polymerase Publishing Resistance Resolution Role Sister Chromatid Exchange Site Structure Therapeutic Work cancer therapy cell growth contextual factors effectiveness evaluation experimental study fly genetic analysis homologous recombination inhibitor/antagonist insight loss of function mutant next generation sequencing novel p53-binding protein 1 repaired response targeted cancer therapy therapeutic target tool

项目摘要

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.

项目总结/文摘