Mechanism and consequences of GAA repeat-mediated chromosomal fragility in yeast

描述（由申请人提供）：弗里德赖希共济失调（FRDA）是在印欧和北非血统个体中发现的最常见的遗传性共济失调。在大多数情况下，该疾病是由携带抑制FRDA基因表达的扩展GAA/TTC轨迹的两个突变等位基因的遗传引起的。除了FRDA，人类基因组中还有数千个其他GAA基因座具有扩增潜力。GAA膨胀和其他相关不稳定性的分子机制知之甚少。本研究的目的是详细阐明如何扩大弗里德赖希共济失调GAA三核苷酸重复导致染色体脆性和随后的染色体重排，以及脆性对酵母，酿酒酵母的大小变化的影响。这是第一次研究GAA介导的脆弱性及其对真核模式生物基因组完整性的影响。具体目标1是确定由GAA三联体重复序列扩展轨道诱导的染色体脆性的分子机制。将测试GAA重复序列形成三链体DNA的长度和方向依赖性导致复制停滞、随后错配修复复合物攻击二级结构和产生双链断裂的假设。这将通过评估染色体臂丢失和基因扩增试验中的脆性，以及通过检查野生型和突变株中的复制停滞和DSB中间体来实现。具体目标2是表征由GAA介导的断裂引起的总染色体重排的结构组织。重排的染色体将使用轮廓钳均相电场凝胶电泳、Southern分析、微阵列上的比较基因组杂交和分子梳理，然后进行荧光原位杂交进行分析。还将确定重排断点的序列组成。具体目标3是确定脆弱性在产生重复大小变化中的作用。将使用PCR和Southern分析估计有丝分裂和减数分裂期间缺失和扩增的频率。将通过比较野生型和缺乏双链断裂形成或修复的菌株之间的重复大小改变的频率来检验脆性可以刺激长尺寸变异的假设。本研究的结果将提供对在FRDA基因座（在Friedreich共济失调患者中）和人类基因组中其他含GAA基因座处观察到的GAA重复的体细胞和种系不稳定性的机制的见解。此外，这项研究将有助于了解与其他类型的致病序列基序相关的不稳定性。 公众健康相关性：本研究旨在阐明GAA三核苷酸重复不稳定性的分子机制。本研究对于了解重复相关疾病的病因和病理以及确定扩展径迹携带者对致瘤性染色体畸变的易感性具有重要意义。

DESCRIPTION (provided by applicant): Friedreich's ataxia (FRDA) is the most frequent inherited ataxia found in individuals of Indo-European and North African descent. In majority of cases, the disease is caused by inheritance of two mutant alleles carrying expanded GAA/TTC tracks that inhibit FRDA gene expression. Besides FRDA, there are thousands of other GAA loci in the human genome that have potential for the expansions. The molecular mechanisms of GAA expansions and other associated instabilities are poorly understood. The goal of this research is the elucidation in detail of how expanded Friedreich's ataxia GAA trinucleotide repeats lead to chromosomal fragility and subsequent chromosomal rearrangements, as well as the effect of fragility on size variations in yeast, Saccharomyces cerevisiae. This is the first study of GAA-mediated fragility and its consequences on genome integrity in a eukaryotic model organism. The Specific Aim 1 is to determine the molecular mechanisms of chromosomal fragility induced by expanded tracks of GAA triplet repeats. The hypothesis that length- and orientation-dependent formation of triplex DNA by GAA repeats leads to replication arrest, subsequent attack of the secondary structure by the mismatch repair complex and generation of double-strand break will be tested. It will be achieved by assessing fragility in chromosomal arm loss and gene amplification assays, and by examining replication arrest and DSB intermediates in wild type and mutant strains. The Specific Aim 2 is to characterize the structural organization of gross chromosomal rearrangements resulting from GAA-mediated breakage. The rearranged chromosomes will be analyzed using contour-clamp homogeneous electric-field gel electrophoresis, Southern analysis, comparative genomic hybridization on microarrays and molecular combing followed by fluorescent in-situ hybridization. The sequence composition of the rearrangement breakpoints will be also determined. The Specific aim 3 is to establish the role of the fragility in the generation of repeat-size variations. The frequencies of deletions and expansions during mitotic and meiotic divisions will be estimated by using PCR and Southern analyses. The hypothesis that fragility can stimulate long size variations will be tested by comparing the frequencies of repeat size alterations between wild type and strains that are deficient in double-strand break formation or repair. The results of this research will provide the insights into mechanisms of somatic and germline instabilities of GAA repeats observed at the FRDA locus (in Friedreich's ataxia patients) and at other GAA- containing loci in the human genome. In addition, this study will contribute to the understanding of instabilities associated with other types of disease-causing sequence motifs. PUBLIV HEALTH RELEVANCE: This research aims to elucidate the molecular mechanisms underlying GAA trinucleotide repeat instability. This study is important for understanding the etiology and pathology of the repeat-associated disorders as well as for determining the susceptibility of the carriers with expanded tracks to the tumorigenic chromosome aberrations.