Nucleotide misincorporation on DNA templates containing N-(deoxyguanosin-N2-yl)-2-(acetylamino)fluorene.

An octadecadeoxynucleotide, modified site-specifically with N-(deoxyguanosin-N2-yl)-2-(acetylamino)fluorene (dG-N2-AAF), was prepared by enzymatic synthesis from a comparably modified decamer and then used as a DNA template in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I containing (exo+) or lacking (exo-) 3'-->5' exonuclease activity. Using exo- Klenow fragment and all four deoxynucleotide triphosphate (dNTPs), primer extension is blocked one base before and opposite dG-N2-AAF. A small fraction of the reaction product represents translesional synthesis, in which dAMP is incorporated opposite the lesion. Kinetic studies of base insertion and chain extension indicate that the frequency of dAMP insertion opposite dG-N2-AAF is higher than that of other deoxynucleotide monophosphates (dNMPs) and of N-(deoxyguanosin-8-yl)-2-(acetylamino)-fluorene (dG-C8-AAF); however, the rate of extension of dA.dG-N2-AAF from the 3' terminus was much lower than that of dA.dG-C8-AAF. We conclude that dG-N2-AAF is a miscoding lesion and capable of generating G-->T transversion mutations in cells.