Comparison of the In Vitro Replication of the 7-(2-Oxoheptyl)-1N2-etheno-2′-deoxyguanosine and 1N2-Etheno-2′-deoxyguanosine Lesions by Sulfolobus solfataricus P2 DNA Polymerase IV (Dpo4)

摘要

Oligonucleotides were synthesized containing the 7-(2-oxoheptyl)-etheno-dGuo adduct, which is derived from the reaction of dGuo and the lipid peroxidation product 4-oxo-2-nonenal. The in vitro replication of 7-(2-oxoheptyl)-etheno-dGuo by the model Y-family polymerase Sulfolobus solfataricus P2 DNA Polymerase IV (Dpo4) was examined in two sequences. The extension products were sequenced using an improved LC-ESI-MS/MS protocol developed in our laboratories and the results were compared to that of the 1,N²-etheno-dGuo adduct in the same sequence contexts. Both etheno adducts were highly miscoding when situated in a 5′-T>XG-3′ local sequence contexts with <4% of the extension products being derived from error-free bypass. The major extension products resulted from the misinsertion of Ade opposite the adduct and a one-base deletion. The major extension products from replication of the etheno lesions in a 5′-C>XG-3′ local sequence context were the result of misinsertion of Ade, a one-base deletion, and error-free bypass. Other minor extension products were also identified. The 7-(2-oxoheptyl)-etheno-dGuo lesion resulted in a larger frequency of misinsertion of Ade, whereas the 1,N²-etheno-dGuo gave more of the one-base deletion product. Conformational studies of duplex DNA containing the 7-(2-oxoheptyl)-etheno-dGuo in a 5′-T>XG-3′ sequence context by NMR indicated the presence of a pH-dependent conformational transition, likely involving the glycosyl bond at the adducted guanosine; the pKa for this transition was lower than that observed for the 1,N²-ε-dGuo lesion. However, the 7-(2-oxoheptyl)-etheno-dGuo lesion, the complementary Cyt, and both flanking base pairs remained disordered at all pH values, which is attributed to the presence of the hydrophobic heptyl group of the 7-(2-oxoheptyl)-etheno-dGuo lesion. The altered pKa value and the structural disorder at the 7-(2-oxoheptyl)-etheno-dGuo lesion site, as compared to the same sequence containing the 1,N²-etheno-dGuo, may contribute to higher frequency of misinsertion of Ade.