DNA repair mechanisms in trinucleotide repeat instability

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序列（TNR）稳定性的机制。TNR序列的扩展，例如，（CAG）n和（CTG）n与某些人类神经和神经变性疾病的进展密切相关，所述疾病包括亨廷顿病（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重复序列分别在发夹茎中形成具有多个A-A和T-T错配的热稳定发夹，因此已经提出DNA错配修复（MMR）和TNR发夹修复在TNR维持中起主要作用。令人惊讶的是，先前在转基因小鼠中的研究表明，错配识别蛋白MSH 2-MSH 3异源二聚体（也称为MutS 2）通过结合（CAG）n形成的发夹并抑制其修复来促进（CAG）n扩增。我们最近的研究表明，人类细胞以切口定向的PCNA依赖性方式催化（CAG）25和（CTG）25发夹的无错误修复。修复靶向切口链在重复序列处的切口，随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，MutS 2不抑制、刺激（CAG）25或（CTG）25发夹修复。有趣的是，我们的初步研究表明，来自HD患者的细胞系在（CAG）n发夹修复中有缺陷，暗示了HD的可能发病机制。在本申请中，具体目标1是评估MMR系统促进（CAG）n扩张的模型。将检查具有或不具有MutS 2过表达的人和动物细胞系在体外修复（CAG）n发夹和在体内复制CAG重复的能力。这些细胞中TNR发夹修复活性和（CAG）n稳定性的测定将阐明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患者来源的细胞系中的发夹修复能力，进一步验证发夹修复缺陷与TNR疾病相关的假设。具体目的3是纯化和表征（CAG）n发夹修复所需的但在HD细胞系中有缺陷的蛋白质。该研究的成功完成将为深入了解TNR扩展的机制和TNR扩展相关疾病的病因学提供重要的参考。

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.