Mechanisms of Palindrome-Mediated Chromosome Fragility

回文介导的染色体脆性机制

• 批准号: 0818122
• 负责人: Kirill Lobachev
• 金额: $ 72万
• 依托单位: Georgia Tech Research Corporation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0818122&HistoricalAwards=false
• 关键词: Mechanisms; Palindrome; Mediated; Chromosome; Fragility

Chromosomal rearrangements play a major role in the evolution of eukaryotic genomes. The presence of repetitive sequences that can adopt non-canonical DNA structures is one of the factors which can predispose chromosomal regions to instability. Palindromic sequences (inverted repeats with or without a unique sequence between them) that can adopt hairpin or cruciform structures are frequently found in regions that are prone to gross chromosomal rearrangements (GCRs) in somatic and germ cells in different organisms. Direct physical evidence was obtained that double-strand breaks (DSBs) occur at the location of long inverted repeats, a triggering event for the genomic instability. However, the mechanisms by which palindromic sequences lead to chromosomal fragility are largely unknown. The overall goal of this research is to elucidate how palindromic sequences induce DSB and GCRs in the yeast Saccharomyces cerevisiae. With previous NSF support, the investigator demonstrated that the cruciform structure adopted by Alu inverted repeats is a target for a nuclease attack, leading to the formation of hairpin-capped breaks that lead to translocations, deletions and gene amplifications. Mutations in several genes that affect palindrome-mediated fragility were identified in preliminary studies. The following specific objectives will make possible in-depth analyses of the key players in cruciform resolution. Objective #1: To determine the role of DNA replication and the post-replicative repair pathway in fragility. Objective #2: To assess the role of Rvb2 and Rvb1 helicases in cruciform metabolism. Objective #3: To use genome-wide screening to identify mutants that are defective in, and prone to, DSB formation at the location of quasi-palindromes. Sensitive biological assays for monitoring DSB formation as well as direct physical analyses of structural intermediates will be used to achieve these objectives. The information resulting from this research will yield important insights into the causes of genome instability at fragile sites in eukaryotic chromosomes. In addition, this work will shed light on the structural organization and evolution of genomes that contain repetitive DNA. This research relies heavily on the active participation of undergraduate and graduate students. In addition, some of these studies will overlap with laboratory activities in an Honors Genetics course taught by the PI at the Georgia Institute of Technology. As in prior NSF-funded studies, the laboratory will continue to recruit female researchers, minority students, and high school teachers. This project will involve collaboration with other laboratories at Georgia Tech, Duke, and Columbia. The results of this work will be presented at various national and international scientific meetings.

染色体重排在真核生物基因组进化中起着重要作用。可以采用非规范DNA结构的重复序列的存在是导致染色体区域不稳定的因素之一。回文序列(带有或不带有唯一序列的反向重复序列)可以采用发夹或十字形结构，经常在不同生物的体细胞和生殖细胞中容易发生大染色体重排(GCR)的区域发现。直接的物理证据表明，双链断裂(DSB)发生在长反向重复序列的位置，这是基因组不稳定的触发事件。然而，回文序列导致染色体脆性的机制在很大程度上是未知的。本研究的总体目标是阐明回文序列如何在酿酒酵母中诱导DSB和GCRs。在之前NSF的支持下，研究人员证明了Alu反向重复序列采用的十字形结构是核酸酶攻击的目标，导致形成发夹状断裂，导致易位、缺失和基因扩增。在初步研究中发现了几个影响回文介导的脆性的基因突变。以下具体目标将使深入分析十字形解决方案的关键因素成为可能。目的#1：确定DNA复制和复制后修复途径在脆性中的作用。目的#2：探讨Rvb2和Rvb1螺旋酶在十字形代谢中的作用。目标#3：使用全基因组筛选来鉴定在准回文位置有缺陷且容易形成DSB的突变体。为实现这些目标，将使用监测DSB形成的敏感生物分析以及结构中间体的直接物理分析。这项研究产生的信息将对真核染色体脆弱部位基因组不稳定的原因产生重要的见解。此外，这项工作将揭示包含重复DNA的基因组的结构组织和进化。这项研究在很大程度上依赖于本科生和研究生的积极参与。此外，其中一些研究将与佐治亚理工学院PI教授的荣誉遗传学课程的实验室活动重叠。与之前由NSF资助的研究一样，该实验室将继续招募女性研究人员、少数族裔学生和高中教师。该项目将涉及与佐治亚理工学院、杜克大学和哥伦比亚大学的其他实验室合作。这项工作的成果将在各种国家和国际科学会议上介绍。