Human DNA polymerase epsilon is a source of C>T mutations at CpG dinucleotides

C to T transitions in CpG dinucleotides are the most prevalent mutations in human cancers and genetic diseases. This bias has been attributed to deamination of 5-methylcytosine (5mC), an epigenetic modification found on CpGs. We recently linked CpG>TpG mutations to replication and hypothesised that errors introduced by Pol epsilon 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 epsilon mutant (P286R) produces an excess of CpG>TpG errors, phenocopying the mutation spectrum of tumours carrying this mutation and deficiencies in mismatch repair. Strikingly, we also discover that wild type Pol epsilon has a 7-fold 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.