SYNTHESIS AND MISCODING SPECIFICITY OF OLIGODEOXYNUCLEOTIDE CONTAINING 8-PHENYL-2'-DEOXYGUANOSINE

Aryl radicals and arenediazonium ions are suspected to react with cell ular DNA, resulting in C8-arylguanine adducts. 8-Phenyl-2'-deoxyguanos ine (8-PhdG) was synthesized as a model adduct by reacting dG with ben zenediazonium chloride and incorporated into oligodeoxynucleotides usi ng phosphoramidite techniques. A site-specifically modified oligodeoxy nucleotide 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 pol ymerase I and mammalian DNA polymerase alpha (pol alpha). Although pri mer 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 catal yzed by pol alpha were strongly blocked opposite the lesion. The fully extended products formed during DNA synthesis were analyzed to quanti fy the miscoding specificities of 8-PhdG. The exo Klenow fragment inco rporated 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 op posite the lesion. When pol alpha was used, 8-PhdG promoted small amou nts of misincorporation of dAMP and dGMP as well as one-and two-base d eletions. The duplex containing 8-PhdG.dG was thermally and thermodyna mically more stable than dG.dG. The duplex containing 8-PhdG.dA was th ermodynamically more stable than dG.dA. We conclude that 8-PhdG is a w eak miscoding lesion, capable of generating G-->T and G-->C transversi ons and deletions in cells.