Synthesis and miscoding specificity of oligodeoxynucleotide containing 8-phenyl-2'-deoxyguanosine.

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 alpha (pol alpha). 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 alpha 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 alpha 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.