Synthesis and Miscoding Specificity of Oligodeoxynucleotide Containing 8-Phenyl-2‘-deoxyguanosine

Abstract

Aryl radicals and arenediazonium ions are suspected to react with cellular DNA, resulting in C8-arylguanine adducts. 8-Phenyl-2‘-deoxyguanosine (8-PhdG) was synthesized as a model adduct by reacting dG with benzenediazonium chloride and incorporated into oligodeoxynucleotides using phosphoramidite techniques. A site-specifically modified oligodeoxynucleotide containing a single 8-PhdG was then used as a template for primer extension reactions catalyzed by the intact (exo⁺) or 3‘→5‘ exonuclease-free (exo^-) Klenow fragment of Escherichia coli DNA polymerase I and mammalian DNA polymerase α (pol α). Although primer extensions catalyzed by the Klenow fragments were retarded at the position of 8-PhdG, most of the primer extension passed the lesion to form the fully extended products. In contrast, primer extensions catalyzed by pol α were strongly blocked opposite the lesion. The fully extended products formed during DNA synthesis were analyzed to quantify the miscoding specificities of 8-PhdG. The exo^- Klenow fragment incorporated primarily dCMP, the correct base, opposite 8-PhdG, along with small amounts of incorporation of dAMP. Two-base deletions were also observed. In contrast, the exo⁺ Klenow fragment incorporated dCMP opposite the lesion. When pol α was used, 8-PhdG promoted small amounts of misincorporation of dAMP and dGMP as well as one- and two-base deletions. The duplex containing 8-PhdG·dG was thermally and thermodynamically more stable than dG·dG. The duplex containing 8-PhdG·dA was thermodynamically more stable than dG·dA. We conclude that 8-PhdG is a weak miscoding lesion, capable of generating G → T and G → C transversions and deletions in cells.