Department of Biology, Tufts University, Medford, MA 02155.
Department of Biology, Tufts University, Medford, MA 02155;
Proc Natl Acad Sci U S A. 2017 Oct 3;114(40):E8392-E8401. doi: 10.1073/pnas.1711283114. Epub 2017 Sep 18.
CAG/CTG repeats are structure-forming repetitive DNA sequences, and expansion beyond a threshold of ∼35 CAG repeats is the cause of several human diseases. Expanded CAG repeats are prone to breakage, and repair of the breaks can cause repeat contractions and expansions. In this study, we found that cotranscriptional R-loops formed at a CAG-70 repeat inserted into a yeast chromosome. R-loops were further elevated upon deletion of yeast RNaseH genes and caused repeat fragility. A significant increase in CAG repeat contractions was also observed, consistent with previous human cell studies. Deletion of yeast cytosine deaminase Fcy1 significantly decreased the rate of CAG repeat fragility and contractions in the background, indicating that Fcy1-mediated deamination is one cause of breakage and contractions in the presence of R-loops. Furthermore, base excision repair (BER) is responsible for causing CAG repeat contractions downstream of Fcy1, but not fragility. The Rad1/XPF and Rad2/XPG nucleases were also important in protecting against contractions, but through BER rather than nucleotide excision repair. Surprisingly, the MutLγ (Mlh1/Mlh3) endonuclease caused R-loop-dependent CAG fragility, defining an alternative function for this complex. These findings provide evidence that breakage at expanded CAG repeats occurs due to R-loop formation and reveal two mechanisms for CAG repeat instability: one mediated by cytosine deamination of DNA engaged in R-loops and the other by MutLγ cleavage. Since disease-causing CAG repeats occur in transcribed regions, our results suggest that R-loop-mediated fragility is a mechanism that could cause DNA damage and repeat-length changes in human cells.
CAG/CTG 重复序列是形成结构的重复 DNA 序列，超过约 35 个 CAG 重复序列的扩展是几种人类疾病的原因。扩展的 CAG 重复序列容易断裂，而断裂的修复可能导致重复收缩和扩展。在这项研究中，我们发现 CAG-70 重复序列插入酵母染色体后会形成转录共形成的 R 环。当缺失酵母 RNaseH 基因时，R 环进一步升高，导致重复脆弱。还观察到 CAG 重复收缩的显著增加，与以前的人类细胞研究一致。在背景中，酵母胞嘧啶脱氨酶 Fcy1 的缺失显著降低了 CAG 重复脆弱性和收缩的速率，表明 Fcy1 介导的脱氨作用是 R 环存在时断裂和收缩的一个原因。此外，碱基切除修复（BER）负责导致 Fcy1 下游的 CAG 重复收缩，但不是脆弱性。Rad1/XPF 和 Rad2/XPG 核酸内切酶在防止收缩方面也很重要，但通过 BER 而不是核苷酸切除修复。令人惊讶的是，MutLγ（Mlh1/Mlh3）内切酶导致 R 环依赖性 CAG 脆弱性，定义了该复合物的另一种功能。这些发现为扩展的 CAG 重复序列断裂是由于 R 环形成而发生提供了证据，并揭示了 CAG 重复不稳定性的两种机制：一种是由参与 R 环的 DNA 的胞嘧啶脱氨作用介导，另一种是由 MutLγ 切割介导。由于致病的 CAG 重复序列发生在转录区域，我们的结果表明 R 环介导的脆弱性是一种可能导致人类细胞中 DNA 损伤和重复长度变化的机制。
