Stability & Fragility of Trinucleotide Repeats in Yeast

稳定

• 批准号: 6730522
• 负责人: CATHERINE H FREUDENREICH
• 金额: $ 23.25万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6730522
• 关键词: DNA damage; DNA replication; Saccharomyces cerevisiae; artificial chromosomes; chemical stability; fungal genetics; genetic screening; mutant; nucleic acid repetitive sequence; polymerase chain reaction

DESCRIPTION (Verbatim from the applicant's abstract): Expansion of trinucleotide repeat (TNR) sequences is the causative mutation for a number of hereditary diseases, including myotonic dystrophy, the most common dystrophy in adults, Fragile X syndrome, the most common form of inherited mental retardation, and neurodegenerative diseases such as Huntington's and the spinocerebellar ataxias. The mechanism of TNR instability is interesting both for understanding the etiology and inheritance of the triplet repeat diseases, and for a basic understanding of genome stability in humans. In addition, expanded CGG/CCG and CTGICAG sequences are sites of chromosome fragility, areas prone to breakage in vivo. Chromosome breakage is implicated in the generation of translocations and deletions found in many types of cancer. The aim of this proposal is to elucidate the mechanisms involved in TNR instability and fragility, and determine how these two unusual characteristics are interrelated using Saccharomyces cerevisiae. A novel genetic assay has been developed that produces a selectable phenotype when a TNR tract expands or breaks. This assay will be used to screen for proteins whose over-expression influences TNR expansion or fragility. The proteins found to influence TNRs will be characterized to determine both their normal cellular functions and their influence on repeat maintenance. In addition, the hypothesis that TNR expansions occur by aberrant lagging strand replication will be tested by analyzing tract stability (by PCR) and fragility (by genetic and physical analysis) in specific yeast replication mutants. The role of the G2IM checkpoint in detecting TNR tract damage and preventing chromosome breakage will be investigated by comparing rates of TNR tract breakage in wild-type and cheokpoint-defective cells. Lastly, these analyses will be extended to other types of minisatellite sequences that act as fragile sites in human cells. The proposed experiments are designed to elucidate not only how simple repeats expand to cause human disease, but also the consequences of and cellular response to expanded tracts, with the goal of understanding how genomic instability can affect human health.

描述（申请人摘要的逐字记录）：扩展 三核苷酸重复（TNR）序列是许多基因的致病突变 遗传性疾病，包括强直性肌营养不良，这是最常见的营养不良 成人脆性 X 综合征是遗传性精神疾病最常见的形式 发育迟缓和神经退行性疾病，例如亨廷顿舞蹈症和 脊髓小脑共济失调。 TNR 不稳定的机制很有趣 为了了解三联体重复疾病的病因和遗传， 以及对人类基因组稳定性的基本了解。此外， 扩展的 CGG/CCG 和 CTGICAG 序列是染色体脆性位点、区域 体内容易破裂。染色体断裂与世代有关 在许多类型的癌症中发现的易位和缺失。此举的目的 提案旨在阐明 TNR 不稳定所涉及的机制以及 脆弱性，并确定这两个不寻常的特征如何相互关联 使用酿酒酵母。一种新的基因检测方法已被开发出来 当 TNR 束扩张或断裂时，会产生可选择的表型。本次检测 将用于筛选过度表达影响 TNR 的蛋白质 扩张或脆弱。被发现影响 TNR 的蛋白质将是 确定其正常细胞功能及其 影响重复维护。此外，假设 TNR 由异常滞后链复制引起的扩增将通过以下方式进行测试 分析管道稳定性（通过 PCR）和脆弱性（通过遗传和物理） 分析）在特定的酵母复制突变体中。 G2IM 的作用 检查点检测 TNR 束损伤并防止染色体断裂 将通过比较野生型和 连接点缺陷的细胞。最后，这些分析将扩展到其他领域 在人类细胞中充当脆弱位点的小卫星序列类型。这 所提出的实验不仅旨在阐明简单的重复 扩大到导致人类疾病，以及细胞的后果 对扩展束的反应，目的是了解基因组如何 不稳定会影响人类健康。