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：
9926844
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 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9926844
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 Effectiveness 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 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.

项目摘要/摘要染色体断裂的修复对于正常细胞生长至关重要，预防并促进癌症 - 引起基因组重排，并确定许多癌症疗法的有效性。在最近的工作中我们的小组在修复染色体断裂的途径中实现了DNA聚合酶theta，有利于短暂的序列重复序列（微观理学）。在AIM 1中，我们将概述Theta介导的结束连接的步骤（TMEJ） - 其分子机制 - 并确定每个步骤的要求。我们将采用组合定义的外染色体底物，靶向染色体断裂的修复分析和定量评估对细胞在各个步骤中有缺陷时对细胞生长和生存能力的影响的评估。在目标2中，我们将澄清theta介导的结束与修复染色体的另一种途径之间的关系休息，同源重组。具体而言，我们将研究BRCA1和53BP1中的缺陷如何，基因参与TMEJ和同源重组的早期步骤，影响通过这两个途径维修。然后，我们还将解决TMEJ途径的重要性霍利迪交界处分辨率，这是同源重组途径的较晚步骤。我们将利用染色体末端结构和修复的测定法，以及通过使用的遗传工具无脊椎动物模型D. melanogaster。在AIM 3中，我们将系统地寻找与聚合酶theta对于一般细胞活力和对电离辐射的耐药性很重要。我们将采用基于慢病毒矢量的CRISPR库，该库策划了专注于310个基因的子集与DNA损伤反应有关。拟议的实验将谈到一个重要但尚未的重要性了解途径不良，尤其是何时以及为什么代表各种方法的最佳解决方案在修复染色体断裂过程中遇到的问题。