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

全基因组分析识别修复 DNA 链断裂所需的基因

基本信息

批准号：
8289252
负责人：
Lysle Kevin Lewis
金额：
$ 29.26万
依托单位：
TEXAS STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8289252
关键词：
Affect Age Aneuploidy Biological Assay Bleomycin Cells Chemicals Chromosomal Breaks Chromosomal Instability Complex DNA DNA Double Strand Break DNA Maintenance DNA Repair DNA Repair Gene DNA Repair Pathway DNA Sequence DNA Sequence Rearrangement DNA damage checkpoint DNA lesion DNA strand break Defect Deoxyribonuclease EcoRI Development Direct Repeats Disease Double Strand Break Repair Etiology Eukaryota Eukaryotic Cell Exhibits Exposure to Gamma Rays Gene Mutation Gene Targeting Genes Genetic Genetic Epistasis Genetic Recombination Genetic Screening Genome Goals HO nuclease Health Hereditary Disease Homologous Gene Human Human Genetics Human Genome Individual Lesion Libraries Link Maintenance Malignant Neoplasms Mating Types Metabolic Pathway Methodology Methods Methyl Methanesulfonate Modeling Mutation Organism Orthologous Gene Pathway Analysis Pathway interactions Phenotype Premature aging syndrome Proteins Radiation induced damage Resistance Role Saccharomyces cerevisiae Saccharomycetales Screening procedure Site Source System Testing Work Yeasts abstracting carcinogenesis chromosome mutation design endonuclease gene function gene repair genome-wide genome-wide analysis homologous recombination improved in vivo mutant repaired research study statistics

项目摘要

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.

描述（由申请人提供）：项目摘要/摘要在称为 DNA 双链断裂 (DSB) 的位点重新连接断裂染色体末端的细胞蛋白至关重要，因为此类损伤修复效率低下会导致突变和染色体不稳定。几种人类遗传疾病与这种类型的 DNA 修复缺陷有关，并已被证明会使受影响的个体容易患上癌症和/或过早衰老。这项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 序列完整性的两条主要途径中的作用。我们独特的基因筛选方法采用两个大型突变菌株文库，在模型真核生物酿酒酵母（芽殖酵母）中鉴定出了修复 DSB 所需的新基因。每个新突变体都缺乏修复 DSB 的能力，这些 DSB 是由细胞内合成 DNA 链断裂核酸内切酶以及暴露于链断裂化学物质后诱导的。这些表型是 DSB 修复突变体的标志，实际上在基因搜索中检测到了 21 个已知的修复基因。总共鉴定出 44 个新基因，这些基因以前未与 DSB 修复相关，但可能在酵母细胞中具有重要功能，并且通过具有同等功能的保守基因，在高等生物体的细胞中具有重要功能。该提案的目的中提出的实验旨在定义这些基因在两种已知的 DSB 修复途径中的功能，这两种途径称为同源重组和非同源末端连接（NHEJ）。 DNA 修复和 DNA 突变率将使用为此目的开发的基因检测进行定量。每个实验都旨在使用相对快速、高通量的方法评估新基因的功能，这些方法保留了产生强大统计数据的能力。研究结果将重点关注那些对维持最关键的特定基因基因组完整性的影响，并且最令人关注的是它们对包括人类在内的其他生物体 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 免遭突变和重排的基因尚未确定。拟议的工作将研究最近被证明可以保护 DNA 完整性的 40 多个酵母基因的功能。大多数酵母保护基因与人类基因具有很强的序列相似性，可能具有相似的功能。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Identification of RNase-resistant RNAs in Saccharomyces cerevisiae extracts: Separation from chromosomal DNA by selective precipitation.

酿酒酵母提取物中 RNase 抗性 RNA 的鉴定：通过选择性沉淀从染色体 DNA 中分离。

DOI：
10.1016/j.ab.2015.09.017
发表时间：
2016
期刊：
Analytical biochemistry
影响因子：
2.9
作者：
Rodriguez,BlancaV;Malczewskyj,EricT;Cabiya,JoshuaM;Lewis,LKevin;Maeder,Corina
通讯作者：
Maeder,Corina

Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells.

提高大肠杆菌细胞中低拷贝数和单拷贝数质粒 DNA 的产量。

DOI：
10.1016/j.mimet.2016.12.016
发表时间：
2017
期刊：
Journal of microbiological methods
影响因子：
2.2
作者：
Wood,WhitneyN;Smith,KyleD;Ream,JenniferA;Lewis,LKevin
通讯作者：
Lewis,LKevin

A multistep genomic screen identifies new genes required for repair of DNA double-strand breaks in Saccharomyces cerevisiae.

DOI：
10.1186/1471-2164-14-251
发表时间：
2013-04-15
期刊：
BMC genomics
影响因子：
4.4
作者：
McKinney JS;Sethi S;Tripp JD;Nguyen TN;Sanderson BA;Westmoreland JW;Resnick MA;Lewis LK
通讯作者：
Lewis LK

Horizontal Agarose Gel Mobility Shift Assay for Protein-RNA Complexes.

蛋白质-RNA 复合物的水平琼脂糖凝胶迁移率变化测定。