Replication studies of carboxymethylated DNA lesions in human cells

Metabolic activation of some N-nitroso compounds (NOCs), an important class of DNA damaging agents, can induce the carboxymethylation of nucleobases in DNA. Very little was previously known about how the carboxymethylated DNA lesions perturb DNA replication in human cells. Here, we investigated the effects of five carboxymethylated DNA lesions, i.e. O-6-CMdG, N-6-CMdA, N-4-CMdC, N3-CMdT and O-4-CMdT on the efficiency and fidelity of DNA replication in HEK293T human embryonic kidney cells. We found that, while neither N-6-CMdA nor N-4-CMdC blocked DNA replication or induced mutations, N3-CMdT, O-4-CMdT and O-6-CMdG moderately blocked DNA replication and induced substantial frequencies of T -> A (81%), T -> C (68%) and G -> A (6.4%) mutations, respectively. In addition, our results revealed that CRISPR-Cas9-mediated depletion of Pol eta resulted in significant drops in bypass efficiencies of N-4-CMdC and N3-CMdT. Diminution in bypass efficiencies was also observed for N-6-CMdA and O-6-CMdG upon depletion of Pol kappa, and for O-6-CMdG upon removal of Pol zeta. Together, our study provided molecular-level insights into the impacts of the carboxymethylated DNA lesions on DNA replication in human cells, revealed the roles of individual translesion synthesis DNA polymerases in bypassing these lesions, and suggested the contributions of O-6-CMdG, N3-CMdT and O-4-CMdT to the mutations found in p53 gene of human gastrointestinal cancers.