Genome-wide analysis identifies genes required for repair of DNA strand breaks

描述(由申请人提供)： 项目摘要/摘要细胞蛋白质在被称为DNA双链断裂(DSB)的位置重新连接断裂染色体的末端是至关重要的，因为这种损伤的低效修复会导致突变和染色体不稳定。一些人类遗传疾病与这种类型的DNA修复缺陷有关，并已被证明容易使受影响的人患上癌症和/或过早衰老。这项拟议工作的目标是提高我们对有效修复断裂DNA所需的基因和代谢途径的理解。具体目标是研究在全基因组遗传搜索中发现的新基因，以确定它们在负责修复DSB和维持DNA序列完整性的两个主要途径中的作用。我们独特的遗传筛选方法，利用两个大型突变菌株文库，已经在模式真核生物酿酒酵母(发芽酵母)中发现了修复DSB所需的新基因。每个新的突变体都缺乏修复DSB的能力，这些DSB是由细胞内合成的DNA断链核酸内切酶和暴露于断链化学物质后诱导的。这些表型是DSB修复突变体的特征，确实在遗传搜索中发现了21个已知的修复基因。共有44个新基因被鉴定出来，这些基因以前没有与DSB修复相关，可能在酵母细胞中具有重要功能，并通过具有相同功能的保守基因在高等生物的细胞中具有重要功能。本方案中提出的实验旨在确定这些基因在两条已知的DSB修复途径中的功能，这两条途径被称为同源重组和非同源末端连接(NHEJ)。DNA修复和DNA突变率将使用为这些目的开发的遗传分析进行量化。每一项实验都旨在使用相对快速、高通量的方法来评估新基因的功能，这些方法保留了产生强大统计数据的能力。这些发现将突出那些对维持生命最关键的特定基因 最令人关切的是它们对包括人类在内的其他生物的DNA稳定性的潜在影响。 公共卫生相关性： 项目叙述许多保护人类细胞内DNA免受突变和重排影响的基因尚未确定。这项拟议的工作将调查最近被证明保护DNA完整性的40多个酵母基因的功能。大多数具有保护性的酵母基因与人类基因有很强的序列相似性，可能具有相似的功能。

DESCRIPTION (provided by applicant): Project Summary/Abstract Cellular proteins that rejoin the ends of broken chromosomes at sites called DNA double-strand breaks (DSBs) are vital because inefficient repair of such lesions leads to mutations and chromosome instability. Several human genetic disorders have been linked to defects in this type of DNA repair and have been shown to predispose affected individuals to development of cancer and/or premature aging. The goal of the proposed work is to improve our understanding of the genes and metabolic pathways required for efficient repair of broken DNA. Specific objectives are to investigate new genes identified in a genome-wide genetic search to determine their roles in the two major pathways responsible for repairing DSBs and in maintenance of DNA sequence integrity. Our unique genetic screening approach, employing two large libraries of mutant strains, has identified new genes required for repair of DSBs in the model eukaryote Saccharomyces cerevisiae (budding yeast). Each of the new mutants lacks the ability to repair DSBs induced by synthesis of a DNA strand-breaking endonuclease inside cells and after exposure to strand-breaking chemicals. These phenotypes are hallmarks of DSB repair mutants and indeed 21 known repair genes were detected in the genetic search. A total of 44 new genes were identified that have not previously been linked to DSB repair and are likely to have important functions in yeast cells and, via conserved genes with equivalent functions, in cells of higher organisms. The experiments proposed in the Aims of this proposal are designed to define the functions of these genes in the two known DSB repair pathways, which are called homologous recombination and nonhomologous end-joining (NHEJ). DNA repair and DNA mutation rates will be quantitated using genetic assays developed for those purposes. Each of the experiments has been designed to assess the functions of the new genes using relatively rapid, high throughput methods that retain the ability to yield strong statistics. The findings will spotlight those specific genes that are most critical for maintenance of genome integrity and are of greatest concern for their potential impact on DNA stability in other organisms including humans. PUBLIC HEALTH RELEVANCE: Project Narrative Many of the genes that protect DNA within human cells from mutations and rearrangements have yet to be identified. The proposed work will investigate the functions of over 40 yeast genes recently shown to protect DNA integrity. Most of the protective yeast genes share strong sequence similarity with human genes which may serve similar functions.