Systematic Genetic Analysis of Yeast NHEJ

描述(申请人提供)：癌症是一种由体细胞中获得的突变引起的遗传病。这种突变背后的一个主要驱动力是细胞衍生过程和外源DNA损伤剂导致的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，因为它会引起几乎普遍存在于癌症中的染色体重排。细胞有两条不同的机制：双链断裂修复、同源定向修复和非同源末端连接(NHEJ)。这项建议的长期目标是了解这两条途径之间的平衡是如何维持的，因此在诱变过程中可能会受到干扰，这首先需要详细了解它们的机制。我们已经开发了专门为研究发芽酵母中的NHEJ而设计的新型遗传系统。它们的基本特征是通过表达的内切酶产生染色体断裂，这样简单的遗传和/或物理分析就可以用来监测断裂的形成和随后的修复。该建议利用这些方法对NHEJ进行系统的突变分析，以实现以下具体目标。在特定的目标#1中，以前的工作被扩展到解决必要的和冗余的蛋白质的潜在参与，我们假设这些蛋白质有很高的参与NHEJ的可能性，特别是SMC蛋白质、染色质修饰复合体和检查点蛋白质。剩下的目标是重点分析酵母NHEJ中已知作用的多功能酶复合体：MREL 1/Rad50/Xrs2，Ku和DNA连接酶IV。特殊目标2通过分离-功能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试图通过在仔细计时的断裂和修复分析中使用物理分析来探索NHEJ中事件的顺序。特定目标#4试图确定NHEJ核心复合体突变体，这些突变体在连接不相容的DNA末端方面存在特定缺陷，假设在招募聚合酶和核酸酶方面存在缺陷。

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.