ABSTRACT:The generation of chemical alkylating agents from nitrosation of glycine and bile acid conjugates in the gastrointestinal tract is hypothesized to initiate carcinogenesis. O 6 -carboxymethylguanine (O 6 -CMG) is a product of DNA alkylation derived from nitrosated glycine. Although the tendency of the structurally related adduct O 6 -methylguanine to code for the misincoporation of TTP during DNA replication is well-established, the impact of the presence of the O 6 -CMG adduct in a DNA template on the efficiency and fidelity of translesion DNA synthesis (TLS) by human DNA polymerases (Pols) have hitherto not been described. Herein, we characterize the ability of the four human TLS Pols η, ι, κ and ζ and the replicative Pol δ to bypass O 6 -CMG in a prevalent mutational hot-spot for cancer. The results indicate that Pol η replicates past O 6 -CMG, incorporating dCMP or dAMP, whereas Pol κ exclusively performs error-free insertion and Pol ι displays the lowest fidelity. Additionally, we found that the subsequent extension step was carried out with high efficiency by TLS Pols η, κ and ζ, while Pol ι was unable to extend from a terminal mismatch. These results provide a first basis of O 6 -CMG-promoted base misincorporation by Y-and B-family polymerases potentially leading to mutational signatures associated with cancer.

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