Systematic Genetic Analysis of Yeast NHEJ

酵母 NHEJ 的系统遗传分析

• 批准号: 7078567
• 负责人: THOMAS EDWARD WILSON
• 金额: $ 22.96万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7078567
• 关键词: DNA repair; Saccharomyces cerevisiae; biological signal transduction; cell cycle proteins; chemical kinetics; chromatin; chromatin immunoprecipitation; enzyme complex; fungal genetics; fungal proteins; gene mutation; genetic recombination; intermolecular interaction; ligase; molecular assembly /self assembly; protein kinase; protein structure function; yeast two hybrid system

DESCRIPTION (provided by applicant): Cancer is a genetic disease caused by mutations acquired in somatic cells. A main driving force behind this mutation is chemical alterations in DNA resulting from cell-derived processes and exogenous DNA damaging agents. A paradox is that the repair systems that normally reverse these DNA lesions must also be responsible for transforming them into heritable sequence alterations. Thus, malignancy is a result of DNA repair failure. The double-stranded chromosome break is a DNA lesion of particular importance as it gives rise to the chromosomal rearrangements that are nearly ubiquitous in cancer. Cells possess two mechanistically distinct pathways of double-strand break repair, homology-directed repair and nonhomologous end joining (NHEJ). The long-term objective of this proposal is to understand how the balance between these two pathways is maintained and might therefore be perturbed during mutagenesis, which first requires a detailed understanding of their mechanisms. We have developed novel genetic systems specifically designed to study NHEJ in budding yeast. Their essential features are creation of a chromosome break by expressed endonucleases in such a way that simple genetic and/or physical analyses can be used to monitor break formation and subsequent repair. This proposal exploits these methodologies to perform a systematic mutational analysis of NHEJ, toward the following specific aims. In Specific Aim #1, previous work is extended to address the potential involvement of essential and redundant proteins that we hypothesize to have a high likelihood of participating in NHEJ, specifically SMC proteins, chromatin modifying complexes and checkpoint proteins. The remaining aims are a focused analysis of the multifunctional enzyme complexes with known roles in yeast NHEJ: Mrel 1/Rad50/Xrs2, Ku and DNA ligase IV. Specific Aim #2 explores the structure-function relationships of these proteins by using separation-offunction analysis to gain insight into their discrete contributions to NHEJ. Specific Aim #3 seeks to explore the sequence of events in NHEJ by using physical analysis in carefully timed break and repair assays. Specific Aim #4 seeks to identify NHEJ core complex mutants that are specifically deficient in joining incompatible DNA ends, hypothesized to be deficient in recruiting polymerases and nucleases.

描述（由申请人提供）：癌症是由在体细胞中获得的突变引起的遗传疾病。该突变背后的主要驱动力是由细胞衍生过程和外源性DNA破坏剂引起的DNA的化学改变。悖论是通常逆转这些DNA病变的修复系统还必须负责将它们转化为可遗传的序列改变。因此，恶性肿瘤是DNA修复衰竭的结果。双链染色体断裂是一种非常重要的DNA病变，因为它引起了几乎无处不在的癌症的染色体重排。细胞具有两种机械上不同的途径，即双链断裂修复，同源指导的修复和非同源末端连接（NHEJ）。该提议的长期目标是了解这两种途径之间的平衡是如何维持的，因此在诱变期间可能会受到干扰，这首先需要对其机制有详细的理解。我们开发了专门研究旨在研究萌芽酵母中NHEJ的新型遗传系统。它们的基本特征是通过表达的核酸酶创建染色体断裂，以使简单的遗传和/或物理分析可用于监测断裂形成和随后的修复。该建议利用这些方法来对NHEJ进行系统的突变分析，以实现以下特定目的。在特定的目标＃1中，以前的工作进行了扩展，以解决我们假设我们假设参与NHEJ的可能性很高的必需和冗余蛋白的潜在参与，特别是SMC蛋白，染色质修饰复合物和检查点蛋白质。其余的目的是对多功能酶复合物的重点分析，在酵母NHEJ中具有已知作用：MREL 1/RAD50/XRS2，KU和DNA连接酶IV。特定的目标＃2通过使用分离函数分析来探索这些蛋白质的结构 - 功能关系，以深入了解其对NHEJ的离散贡献。特定目标＃3试图通过在精心定时的断裂和修复测定中使用物理分析来探索NHEJ中事件的顺序。特定的目标＃4旨在识别NHEJ核心复合物突变体，这些突变体特异性缺乏连接不兼容的DNA末端，认为缺乏募集聚合酶和核酸酶。