Translesion synthesis across polycyclic aromatic hydrocarbon diol epoxide adducts of deoxyadenosine by Sulfolobus solfataricus DNA polymerase Dpo4.

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.