The roles of polymerases ν and θ in the 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) nu and Pol theta in translesion synthesis (TLS) in cells. Here, we examined the functions of both polymerases in bypassing the major-groove O (6)-alkyl-2'-deoxyguanosine (O (6)-alkyl-dG) and the minor-groove N (2)-alkyl-dG lesions in human cells, where the alkyl groups are ethyl (Et), n-butyl (nBu), and, for O (6)-alkyl-dG, pyridyloxobutyl (POB). We found that Pol nu and Pol theta promote TLS across the major-groove O (6)-alkyl-dG lesions. The O (6)-alkyl-dG lesions mainly induced G->A mutations, which were modulated by the two TLS polymerases and the structures of the alkyl groups. Simultaneous ablation of Pol nu and Pol theta resulted in diminished mutation frequencies for all three O (6)-alkyl-dG lesions, depletion of Pol nu alone reduced mutations only for O (6)-nBu-dG, and sole loss of Pol theta attenuated the mutation rates for both O (6)-nBu-dG and O (6)-POB-dG. Replication across the two N (2)-alkyl-dG lesions was error-free, and Pol nu and Pol theta were dispensable for their replicative bypass. Together, our results provide critical knowledge about the involvement of Pol nu and Pol theta in bypassing alkylated guanine lesions in human cells.