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

描述(申请人提供)：Friedreich‘s共济失调(FRDA)是最常见的遗传性共济失调，在印欧和北非后裔中发现。在大多数情况下，这种疾病是由于遗传了两个突变的等位基因，携带抑制FRDA基因表达的扩展的GAA/TTC轨道而引起的。除了FRDA，人类基因组中还有数千个其他GAA基因座具有扩展的潜力。GAA膨胀和其他相关不稳定性的分子机制还知之甚少。这项研究的目的是详细阐明扩展的Friedreich共济失调GAA三核苷酸重复如何导致染色体脆性和随后的染色体重排，以及脆性对酿酒酵母大小变异的影响。这是第一次在真核模式生物中研究GAA介导的脆弱性及其对基因组完整性的影响。具体目的1是确定由GAA三联体重复序列扩展轨迹引起的染色体脆性的分子机制。GAA重复形成的三链DNA的长度和取向相关的假设会导致复制停止，随后错配修复复合体对二级结构的攻击和双链断裂的产生将受到检验。它将通过评估染色体臂丢失和基因扩增分析中的脆弱性，以及通过检查野生型和突变株的复制停滞和DSB中间体来实现。具体目标2是描述由GAA介导的断裂引起的总染色体重排的结构组织。重排后的染色体将通过轮廓钳均一电场凝胶电泳、Southern分析、微阵列比较基因组杂交和分子梳理以及荧光原位杂交等方法进行分析。重排断点的序列组成也将被确定。具体目标3是确定脆弱性在重复大小变异产生中的作用。有丝分裂和减数分裂过程中的缺失和扩展的频率将通过聚合酶链式反应和Southern分析来估计。通过比较野生型和缺乏双链断裂形成或修复缺陷的菌株之间重复大小变化的频率，将检验脆性可以刺激长大小变异的假设。这项研究的结果将为在FRDA基因座(在Friedreich‘s共济失调患者中)和在人类基因组中其他包含GAA的基因座观察到的GAA重复的体细胞和生殖系不稳定性的机制提供洞察力。此外，这项研究将有助于理解与其他类型的致病序列基序相关的不稳定性。PUBLIV健康相关性：这项研究旨在阐明GAA三核苷酸重复不稳定的分子机制。这项研究对于了解重复相关疾病的病因和病理以及确定具有扩展轨迹的携带者对致瘤染色体异常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.