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

• 批准号: 9596072
• 负责人: DALE A RAMSDEN
• 金额: $ 44.26万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 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/9596072
• 关键词: Address; Animal Model; BRCA1 gene; BRCA2 gene; Biological Assay; Breast Cancer Model; CRISPR library; Cancer Etiology; 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; 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

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/摘要 染色体断裂的修复对正常细胞生长至关重要，可以预防和促进癌症- 导致基因组重排，并决定许多癌症治疗的有效性。在最近的工作中 我们的研究小组认为DNA聚合酶theta参与了修复染色体断裂的途径， 微同源性（microhomologies）在目标1中，我们将概述Theta介导的末端连接（TMEJ）的步骤 - 它的分子机制-并确定每个步骤的要求。我们将采用以下组合： 确定的染色体外底物，分析靶向染色体断裂的修复，以及定量 评估当细胞在各个步骤中存在缺陷时对细胞生长和活力的影响。在目标2中， 阐明Theta介导的末端连接与另一条染色体修复途径的关系 断裂同源重组具体来说，我们将研究BRCA 1和53 BP 1基因的缺陷如何影响基因表达。 参与TMEJ和同源重组共同的早期步骤，影响TMEJ和同源重组之间的选择。 通过这两条途径进行修复。然后，我们还将讨论TMEJ途径作为替代途径的重要性 到霍利迪连接分辨率，一个后期步骤特定的同源重组途径。我们将利用 染色体末端结构和修复的测定，以及通过使用 无脊椎动物模型D.黑腹菌在目标3中，我们将系统地寻找新的遗传相互作用， 聚合酶θ对一般细胞活力和细胞对电离辐射的抵抗都很重要。 我们将采用一个基于慢病毒载体的CRISPR文库，该文库经过精心策划，专注于310个基因的子集 与DNA损伤反应有关。拟议的实验将说明一项重要但尚未实现的 人们对途径的工作原理知之甚少，尤其是何时以及为什么它代表了各种问题的最佳解决方案。 修复染色体断裂过程中遇到的问题。