Translesion synthesis DNA polymerases promote error-free replication through the minor-groove DNA adduct 3-deaza-3-methyladenine

N3-Methyladenine (3-MeA) is formed in DNA by reaction with S-adenosylmethionine, the reactive methyl donor, and by reaction with alkylating agents. 3-MeA protrudes into the DNA minor groove and strongly blocks synthesis by replicative DNA polymerases (Pols). However, the mechanisms for replicating through this lesion in human cells remain unidentified. Here we analyzed the roles of translesion synthesis (TLS) Pols in the replication of 3-MeA-damaged DNA in human cells. Because 3-MeA has a short half-life in vitro, we used the stable 3-deaza analog, 3-deaza-3-methyladenine (3-dMeA), which blocks the DNA minor groove similarly to 3-MeA. We found that replication through the 3-dMeA adduct is mediated via three different pathways, dependent upon Pol/Pol, Pol, and Pol. As inferred from biochemical studies, in the Pol/Pol pathway, Pol inserts a nucleotide (nt) opposite 3-dMeA and Pol extends synthesis from the inserted nt. In the Pol pathway, Pol carries out both the insertion and extension steps of TLS opposite 3-dMeA, and in the Pol pathway, Pol extends synthesis following nt insertion by an as yet unidentified Pol. Steady-state kinetic analyses indicated that Pol and Pol insert the correct nt T opposite 3-dMeA with a much reduced catalytic efficiency and that both Pols exhibit a high propensity for inserting a wrong nt opposite this adduct. However, despite their low fidelity of synthesis opposite 3-dMeA, TLS opposite this lesion replicates DNA in a highly error-free manner in human cells. We discuss the implications of these observations for TLS mechanisms in human cells.