Polymerase ι promotes C>T substitutions in smoking models and in the platinum chemotherapy–induced SBS31 mutation signature

DNA damage occasionally leads to mutations even in DNA repair–proficient cells via translesion synthesis (TLS), an efficient but error-prone DNA damage tolerance pathway. In the human genome, there are three closely related Y-family TLS polymerases (η, κ, and ι). Polymerase η is specialized for the error-free replication of UV adducts, while polymerase κ is known to replicate over various heterocyclic DNA adducts. The function of polymerase ι is less clear and this gene is missing from several vertebrate clades, yeast, most birds, and plants. Using wild-type and TLS polymerase ( POLH , POLK , POLI ) knockout human TK6 and RPE-1 cell lines and whole genome sequencing, we associate these polymerases to mutagenesis induced by the common chemotherapy agent cisplatin and the smoke-mimicking compound 4-nitroquinoline-1-oxide (4-NQO). We found that polymerase ι specifically introduces C>T mutations at both cisplatin and 4-NQO lesions, confirmed in two further cell lines. Using non-negative matrix factorization, we derived a polymerase ι–specific base substitution signature, which corresponds to COSMIC cancer mutational signature SBS31 with a cosine similarity of 0.97. By analyzing the mutational spectra in more than 3500 human metastatic tumors, we showed that SBS31 in platinum-treated samples and SBS92 in smoking-related cancers is associated with polymerase ι expression. Our study suggests an in vivo mutagenic function for polymerase ι and highlights its role in tissue-dependent chemotherapy–induced mutagenesis that may contribute to tumor heterogeneity.