The roles of polymerases ν and θ in replicative bypass of O⁶- and N²-alkyl-2′-deoxyguanosine lesions in human cells

Exogenous and endogenous chemicals can react with DNA to produce DNA lesions that may block DNA replication. Not much is known about the roles of polymerase (Pol) ν and Pol θ in translesion synthesis (TLS) in cells. Here we examined the functions of these two polymerases in bypassing major-groove O⁶-alkyl-2′-deoxyguanosine (O⁶-alkyl-dG) and minor-groove N²-alkyl-dG lesions in human cells, where the alkyl groups are ethyl, n-butyl (nBu), and, for O⁶-alkyl-dG, pyridyloxobutyl. We found that Pol ν and Pol θ promote TLS across major-groove O⁶-alkyl-dG lesions. O⁶-alkyl-dG lesions mainly induced G→A mutations that were modulated by the two TLS polymerases and the structures of the alkyl groups. Simultaneous ablation of Pol ν and Pol θ resulted in diminished mutation frequencies for all three O⁶-alkyl-dG lesions. Depletion of Pol ν alone reduced mutations only for O⁶-nBu-dG, and sole loss of Pol θ attenuated the mutation rates for O⁶-nBu-dG and O⁶-pyridyloxobutyl-dG. Replication across the two N²-alkyl-dG lesions was error-free, and Pol ν and Pol θ were dispensable for their replicative bypass. Together, our results provide critical knowledge about the involvement of Pol ν and Pol θ in bypassing alkylated guanine lesions in human cells.