Mechanisms of Palindrome-Mediated Chromosome Fragility

染色体重排在真核生物基因组的进化中起着重要作用。 可采用非规范DNA结构的重复序列的存在是可使染色体区域倾向于不稳定的因素之一。 回文序列（反向重复序列，它们之间有或没有一个独特的序列），可以采用发夹或十字形结构，经常发现在区域，易于总染色体重排（GCR）的体细胞和生殖细胞在不同的生物体。 直接物理证据表明，双链断裂（DSB）发生在长反向重复序列的位置，这是基因组不稳定性的触发事件。 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GCR。 在之前的NSF支持下，研究人员证明了Alu反向重复序列所采用的十字形结构是核酸酶攻击的目标，导致形成发夹帽断裂，导致易位，缺失和基因扩增。 初步研究发现了几个影响回文介导的脆性的基因突变。 以下具体目标将使我们能够深入分析解决十字形问题的主要参与者。 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：评估Rvb2和Rvb1解旋酶在十字形代谢中的作用。 目标三：使用全基因组筛选来鉴定在准回文位置处DSB形成中有缺陷且易于形成的突变体。用于监测DSB形成的灵敏生物测定以及结构中间体的直接物理分析将用于实现这些目标。 从这项研究中得到的信息将产生重要的见解基因组不稳定性的原因在真核细胞染色体的脆弱位点。 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，其中一些研究将与格鲁吉亚理工学院PI教授的荣誉遗传学课程中的实验室活动重叠。 与之前NSF资助的研究一样，该实验室将继续招募女性研究人员，少数民族学生和高中教师。 该项目将涉及与格鲁吉亚理工学院、杜克大学和哥伦比亚大学的其他实验室的合作。 这项工作的结果将在各种国家和国际科学会议上介绍。

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.