DNA repair mechanisms in trinucleotide repeat instability

三核苷酸重复不稳定性中的DNA修复机制

• 批准号: 8469519
• 负责人: Guo-Min Li
• 金额: $ 21.84万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8469519
• 关键词: Animals; Binding; Biochemical; Biological Assay; CAG repeat; Cell Line; Cells; DNA; DNA Repair; DNA biosynthesis; Defect; Disease; Ensure; Etiology; Fragile X Syndrome; Friedreich Ataxia; Generations; Genes; Genetic; Genome; Goals; Hela Cells; Human; Huntington Disease; In Vitro; Lead; Length; Link; MSH2 gene; MSH3 gene; Maintenance; Mismatch Repair; Modeling; Myotonic Dystrophy; Neurodegenerative Disorders; Neuromuscular Diseases; Nuclear Extract; Nucleotides; Pathogenesis; Pathway interactions; Patients; Play; Population; Protein Binding; Proteins; RNA Interference; Role; Site; Surgical incisions; System; Testing; Transgenic Mice; Trinucleotide Repeat Expansion; Trinucleotide Repeats; Work; base; effective therapy; in vivo; insight; nervous system disorder; overexpression; prevent; public health relevance; repaired; research study; screening; stem

DESCRIPTION (provided by applicant): The long-term goal of the project is to understand the mechanisms by which cells maintain trinucleotide repeat (TNR) stability. Expansions of TNR sequences, e.g., (CAG)n and (CTG)n, are tightly associated with progression of certain human neurological and neurodegenerative diseases including Huntington's disease (HD) and myotonic dystrophy. However, the mechanisms and factors that promote/prevent TNR expansions are unknown. Because CAG and CTG repeats form thermo- stable hairpins with multiple A-A and T-T mispairs in the hairpin stem, respectively, DNA mismatch repair (MMR) and TNR hairpin repair have been proposed to play major roles in TNR maintenance. Surprisingly, previous studies in transgenic mice suggest that mismatch recognition protein MSH2- MSH3 heterodimer (also called MutS2) promotes (CAG)n expansions by binding to (CAG)n-formed hairpins and inhibiting their repair. Our recent studies have shown that human cells catalyze error-free repair of (CAG)25 and (CTG)25 hairpins in a nick-directed PCNA-dependent manner. The repair targets the nicked strand for incisions at the repeat sequences, followed by repair DNA synthesis using the continuous strand as a template, thereby ensuring TNR stability. However, MutS2 does not inhibit, stimulates (CAG)25 or (CTG)25 hairpin repair. Interestingly, our preliminary studies have shown that cell lines derived from HD patients are defective in (CAG)n hairpin repair, hinting possible pathogenesis for HD. In this application, Specific Aim 1 is to evaluate the model that the MMR system promotes (CAG)n expansion. Human and animal cell lines with or without MutS2 overexpression will be examined for their ability to repair (CAG)n hairpins in vitro and to replicate CAG repeats in vivo. Determination of TNR hairpin repair activity and (CAG)n stability in these cells will clarify if the MMR system is responsible for (CAG)n expansions in human cells. Specific Aim 2 is to further test the hypothesis that hairpin repair defects are associated with TNR diseases, by screening hairpin repair proficiency in cell lines derived from HD patients. Specific Aim 3 is to purify and characterize a protein required for (CAG)n hairpin repair but defective in an HD cell line. A successful completion of the proposed work will provide significant insight into the mechanisms of TNR expansions and the etiology of TNR expansion-associated diseases.

描述（由申请人提供）：该项目的长期目标是了解细胞维持三核苷酸重复（TNR）稳定性的机制。 TNR 序列的扩展，例如 (CAG)n 和 (CTG)n，与某些人类神经系统和神经退行性疾病的进展密切相关，包括亨廷顿病 (HD) 和强直性肌营养不良。然而，促进/阻止 TNR 扩张的机制和因素尚不清楚。由于 CAG 和 CTG 重复序列分别形成热稳定发夹，并在发夹茎中分别具有多个 A-A 和 T-T 错配，因此 DNA 错配修复 (MMR) 和 TNR 发夹修复已被认为在 TNR 维持中发挥主要作用。令人惊讶的是，之前对转基因小鼠的研究表明，错配识别蛋白 MSH2-MSH3 异二聚体（也称为 MutS2）通过与 (CAG)n 形成的发夹结合并抑制其修复来促进 (CAG)n 扩展。我们最近的研究表明，人类细胞以切口定向 PCNA 依赖性方式催化 (CAG)25 和 (CTG)25 发夹的无差错修复。修复的目标是在重复序列处切割切口链，然后使用连续链作为模板进行修复 DNA 合成，从而确保 TNR 稳定性。然而，MutS2 不会抑制、刺激 (CAG)25 或 (CTG)25 发夹修复。有趣的是，我们的初步研究表明，源自 HD 患者的细胞系在 (CAG)n 发夹修复方面存在缺陷，这暗示了 HD 的可能发病机制。在此应用中，具体目标 1 是评估 MMR 系统促进 (CAG)n 扩展的模型。将检查具有或不具有 MutS2 过表达的人类和动物细胞系在体外修复 (CAG)n 发夹和在体内复制 CAG 重复序列的能力。测定这些细胞中的 TNR 发夹修复活性和 (CAG)n 稳定性将阐明 MMR 系统是否负责人类细胞中的 (CAG)n 扩增。具体目标 2 是通过筛选 HD 患者细胞系的发夹修复能力，进一步检验发夹修复缺陷与 TNR 疾病相关的假设。具体目标 3 是纯化和表征 (CAG)n 发夹修复所需但在 HD 细胞系中存在缺陷的蛋白质。成功完成拟议的工作将为 TNR 扩展的机制和 TNR 扩展相关疾病的病因学提供重要的见解。