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（申请人提供）：癌症是一种由体细胞突变引起的遗传性疾病。这种突变背后的主要驱动力是细胞衍生过程和外源 DNA 损伤剂引起的 DNA 化学变化。一个悖论是，通常逆转这些 DNA 损伤的修复系统也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修复失败的结果。双链染色体断裂是一种特别重要的 DNA 损伤，因为它会引起癌症中几乎普遍存在的染色体重排。细胞拥有双链断裂修复、同源定向修复和非同源末端连接（NHEJ）这两种机制不同的途径。该提案的长期目标是了解这两种途径之间的平衡是如何维持的，以及如何在诱变过程中受到干扰，这首先需要详细了解它们的机制。我们开发了专门用于研究芽殖酵母 NHEJ 的新型遗传系统。它们的基本特征是通过表达的核酸内切酶产生染色体断裂，从而可以使用简单的遗传和/或物理分析来监测断裂形成和随后的修复。该提案利用这些方法对 NHEJ 进行系统的突变分析，以实现以下具体目标。在具体目标 #1 中，之前的工作被扩展至解决必需蛋白和冗余蛋白的潜在参与问题，我们假设这些蛋白很可能参与 NHEJ，特别是 SMC 蛋白、染色质修饰复合物和检查点蛋白。剩下的目标是重点分析在酵母 NHEJ 中具有已知作用的多功能酶复合物：Mrel 1/Rad50/Xrs2、Ku 和 DNA 连接酶 IV。具体目标#2 通过使用功能分离分析来探索这些蛋白质的结构-功能关系，以深入了解它们对 NHEJ 的离散贡献。具体目标#3 旨在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 中的事件顺序。具体目标 #4 旨在鉴定 NHEJ 核心复合体突变体，这些突变体在连接不相容的 DNA 末端方面特别缺乏，假设在招募聚合酶和核酸酶方面存在缺陷。