C-to-T transitions in CpG dinucleotides are the most prevalent mutations in human cancers and genetic diseases. These mutations have been attributed to deamination of 5-methylcytosine (5mC), an epigenetic modification found on CpGs. We recently linked CpG>TpG mutations to replication and hypothesized that errors introduced by polymerase ε (Pol ε) may represent an alternative source of mutations. Here we present a new method called polymerase error rate sequencing (PER-seq) to measure the error spectrum of DNA polymerases in isolation. We find that the most common human cancer-associated Pol ε mutant (P286R) produces an excess of CpG>TpG errors, phenocopying the mutation spectrum of tumors carrying this mutation and deficiencies in mismatch repair. Notably, we also discover that wild-type Pol ε has a sevenfold higher error rate when replicating 5mCpG compared to C in other contexts. Together, our results from PER-seq and human cancers demonstrate that replication errors are a major contributor to CpG>TpG mutagenesis in replicating cells, fundamentally changing our understanding of this important disease-causing mutational mechanism.

CpG 二核苷酸的 C-to-T 转换是人类癌症和遗传疾病中最普遍的突变。这些突变归因于 5-甲基胞嘧啶 （5mC） 的脱氨作用，这是一种在 CpG 上发现的表观遗传修饰。我们最近将 CpG>TpG 突变与复制联系起来，并假设聚合酶ε （Pol ε） 引入的错误可能代表了突变的替代来源。在这里，我们提出了一种称为聚合酶错误率测序 （PER-seq） 的新方法，用于测量分离 DNA 聚合酶的错误谱。我们发现最常见的人类癌症相关 Pol ε 突变体 （P286R） 产生过量的 CpG>TpG 错误，表型复制携带该突变和错配修复缺陷的肿瘤的突变谱。值得注意的是，我们还发现，与其他情况下复制 5mCpG 相比，野生型 Pol ε 的错误率高出 7 倍。总之，我们来自 PER-seq 和人类癌症的结果表明，复制错误是复制细胞中 CpG>TpG 诱变的主要因素，从根本上改变了我们对这一重要致病突变机制的理解。