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The mechanisms by which derivatives of polycyclic aromatic hydrocarbons (PAHs) cause mutations have been of considerable interest. Three different N(6)-adenyl PAH-diol epoxide oligonucleotide derivatives were studied with the archebacterial translesion DNA polymerase Sulfolobus solfataricus Dpo4. Steady-state kinetic analysis indicated insertion of all four dNTPs opposite each of the three N(6)-adenyl PAH adducts, with only slightly varying misincorporation efficiencies. Full-length extension of shorter primers paired with templates containing the N(6)-adenyl PAH derivatives proceeded to apparent completion at 45 degrees C in the presence of added dimethyl sulfoxide. Analysis of the products by high-performance liquid chromatography/collision-induced mass spectrometry indicated the presence of mixtures of products with each PAH adduct. These mixtures correspond to both error-free synthesis and mixtures of polymerization/realignment steps. With an unmodified template, only the expected A:T and G:C pairing was detected in the primer extension products under these conditions, with no frameshifts. These results demonstrate the complexity of polymerization opposite these bulky N(6)-adenyl PAH adducts, even with a single polymerase.