Huntington's disease (HD), a neurodegenerative disease characterized by progressive dementia, psychiatric problems, and chorea, is known to be caused by CAG repeat expansions in the HD gene HTT. However, the mechanism of this pathology is not fully understood. The translesion DNA polymerase θ (Polθ) carries a large insertion sequence in its catalytic domain, which has been shown to allow DNA loop-outs in the primer strand. As a result of high levels of oxidative DNA damage in neural cells and Polθ's subsequent involvement in base excision repair of oxidative DNA damage, we hypothesized that Polθ contributes to CAG repeat expansion while repairing oxidative damage within HTT. Here, we performed Polθ-catalyzed in vitro DNA synthesis using various CAG•CTG repeat DNA substrates that are similar to base excision repair intermediates. We show that Polθ efficiently extends (CAG)n•(CTG)n hairpin primers, resulting in hairpin retention and repeat expansion. Polθ also triggers repeat expansions to pass the threshold for HD when the DNA template contains 35 repeats upward. Strikingly, Polθ depleted of the catalytic insertion fails to induce repeat expansions regardless of primers and templates used, indicating that the insertion sequence is responsible for Polθ's error-causing activity. In addition, the level of chromatin-bound Polθ in HD cells is significantly higher than in non-HD cells and exactly correlates with the degree of CAG repeat expansion, implying Polθ's involvement in triplet repeat instability. Therefore, we have identified Polθ as a potent factor that promotes CAG•CTG repeat expansions in HD and other neurodegenerative disorders.

中文翻译：

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DNA聚合酶θ通过其催化结构域的插入序列促进亨廷顿病中CAG•CTG重复扩增。


亨廷顿病 (HD) 是一种以进行性痴呆、精神问题和舞蹈病为特征的神经退行性疾病，已知是由 HD 基因 HTT 中的 CAG 重复扩增引起的。然而，这种病理机制尚不完全清楚。跨损伤 DNA 聚合酶 θ (Polθ) 在其催化结构域中携带一个大的插入序列，这已被证明允许引物链中的 DNA 环出。由于神经细胞中高水平的氧化性 DNA 损伤以及 Polθ 随后参与氧化性 DNA 损伤的碱基切除修复，我们假设 Polθ 有助于 CAG 重复扩增，同时修复 HTT 内的氧化损伤。在这里，我们使用类似于碱基切除修复中间体的各种CAG•CTG重复DNA底物进行了Polθ催化的体外DNA合成。我们表明，Polθ 有效延伸 (CAG)n•(CTG)n 发夹引物，导致发夹保留和重复扩增。当 DNA 模板包含 35 个以上的重复时，Polθ 还会触发重复扩展以通过 HD 阈值。引人注目的是，无论使用何种引物和模板，催化插入耗尽的 Polθ 都无法诱导重复扩增，这表明插入序列是 Polθ 导致错误的活性的原因。此外，HD细胞中染色质结合的Polθ水平显着高于非HD细胞，并且与CAG重复扩增的程度完全相关，这意味着Polθ参与了三联体重复不稳定。因此，我们发现 Polθ 是在 HD 和其他神经退行性疾病中促进 CAG•CTG 重复扩增的有效因子。