Division of Neurology, Department of Neurosciences, University of California, San Diego, CA 92103, USA.
Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Nucleic Acids Res. 2018 May 4;46(8):4022-4032. doi: 10.1093/nar/gky143.
DNA repeat expansion underlies dozens of progressive neurodegenerative disorders. While the mechanisms driving repeat expansion are not fully understood, increasing evidence suggests a central role for DNA mismatch repair. The mismatch repair recognition complex MutSβ (MSH2-MSH3) that binds mismatched bases and/or insertion/deletion loops has previously been implicated in GAA•TTC, CAG•CTG and CGG•CCG repeat expansion, suggesting a shared mechanism. MutSβ has been studied in a number of models, but the contribution of subsequent steps mediated by the MutL endonuclease in this pathway is less clear. Here we show that MutLγ (MLH1-MLH3) is the MutL complex responsible for GAA•TTC repeat expansion. Lentiviral expression of shRNA targeting MutL nuclease components MLH1, PMS2, and MLH3 revealed that reduced expression of MLH1 or MLH3 reduced the repeat expansion rate in a human Friedreich ataxia cell model, while targeting PMS2 did not. Using splice-switching oligonucleotides we show that MLH3 isoform 1 is active in GAA•TTC repeat expansion while the nuclease-deficient MLH3 isoform 2 is not. MLH3 isoform switching slowed repeat expansion in both model cells and FRDA patient fibroblasts. Our work indicates a specific and active role for MutLγ in the expansion process and reveals plausible targets for disease-modifying therapies.
DNA 重复扩展是数十种进行性神经退行性疾病的基础。虽然导致重复扩展的机制尚未完全了解，但越来越多的证据表明 DNA 错配修复起着核心作用。先前已经表明，MutSβ（MSH2-MSH3）识别复合物结合错配碱基和/或插入/缺失环，与 GAA•TTC、CAG•CTG 和 CGG•CCG 重复扩展有关，表明存在共同的机制。MutSβ 在许多模型中进行了研究，但在该途径中由 MutL 内切酶介导的后续步骤的贡献不太清楚。在这里，我们表明 MutLγ（MLH1-MLH3）是负责 GAA•TTC 重复扩展的 MutL 复合物。针对 MutL 核酸酶成分 MLH1、PMS2 和 MLH3 的 shRNA 的慢病毒表达表明，MLH1 或 MLH3 的表达降低会降低人类弗里德里希共济失调细胞模型中的重复扩展率，而靶向 PMS2 则不会。使用剪接转换寡核苷酸，我们表明 MLH3 同种型 1 在 GAA•TTC 重复扩展中是活跃的，而无核酸酶的 MLH3 同种型 2 则不是。MLH3 同种型转换在两种模型细胞和 FRDA 患者成纤维细胞中均减缓了重复扩展。我们的工作表明 MutLγ 在扩展过程中具有特定的和活跃的作用，并揭示了潜在的疾病修饰治疗靶标。
