Genetics of Yeast Nonhomologous End-Joining

描述（由申请人提供）：癌症是一种由体细胞获得突变引起的遗传性疾病。这种突变背后的主要驱动力是由细胞衍生过程和外源性DNA损伤剂引起的DNA化学改变。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，因为它引起了几乎在癌症中普遍存在的染色体重排。细胞具有双链断裂修复、同源定向修复和非同源末端连接（NHEJ）两种机制不同的途径。这项建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。我们已经开发了新的遗传系统，专门用于研究芽殖酵母中的NHEJ。它们的基本特征是通过表达的内切酶以这样一种方式产生染色体断裂，简单的遗传和/或物理分析可以用来监测断裂的形成和随后的修复。本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Specific Aim #1中，先前的工作扩展到解决必要和冗余蛋白的潜在参与，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。剩下的目标是集中分析在酵母NHEJ中已知作用的多功能酶复合物：Mrel 1/Rad50/Xrs2， Ku和DNA连接酶IV。Specific Aim #2通过使用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旨在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中的事件序列。Specific Aim #4旨在鉴定NHEJ核心复合物突变体，这些突变体在连接不相容的DNA末端方面存在特异性缺陷，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.