Spontaneous mutations in TLS mutant RPE-1 cells. Spontaneous mutagenesis in human cells is controlled by REV1-Polymerase ζ and PRIMPOL

Translesion DNA synthesis (TLS) facilitates DNA replication over damaged or difficult-to-replicate templates by employing specialised DNA polymerases. We investigated the effect on spontaneous mutagenesis of three main TLS control mechanisms: REV1 and PCNA ubiquitylation that recruit TLS polymerases, and PRIMPOL that creates post-replicative gaps. Using whole genome sequencing of cultured human RPE-1 cell clones, we found that REV1 and Polymerase ζ were wholly responsible for one component of base substitution mutagenesis that resembled homologous recombination deficiency, whereas the remaining component that was similar to oxidative mutagenesis was reduced in PRIMPOL–/– cells. Small deletions in short repeats appeared in REV1–/– PCNAK164R/K164R double mutants, revealing an alternative TLS mechanism. Also, 500-5000 bp deletions appeared in REV1–/– mutants, and chromosomal instability was observed in REV1–/– PRIMPOL–/– cells. Our results indicate that TLS protects the genome from deletions and large rearrangements at the expense of being responsible for the majority of spontaneous base substitutions.