2'-Deoxyoxanosine (dOxo) is a novel DNA lesion produced by the reaction of 2'-deoxyguanosine (dGuo) with nitrous acid and nitric oxide [Suzuki, T., Yamaoka, R., Nishi, M., Ide, H., and Makino, K. (1996) J. Am. Chem. Soc. 118, 2515-2516]. In this work, 2'-deoxyoxanosine 5'-triphosphate (dOTP) was prepared by nitrous acid treatment of 2'-deoxyguanosine 5'-triphosphate (dGTP), and its incorporation into DNA by DNA polymerases was investigated to elucidate the substrate and mutagenic properties of dOTP. Primed M13mp18 DNA was replicated by Escherichia coli DNA polymerase I Klenow fragment (Pol I Kf) in the presence of three normal dNTPs and dOTP or 2'-deoxyxanthosine 5'-triphosphate (dXTP), another major product of reaction of dGTP with nitrous acid and nitric oxide. dOTP substituted for dGTP and to a lesser extent for dATP, while dXTP substituted slightly for dGTP but not for dATP. Neither dOTP nor dXTP substituted for dCTP and dTTP. The similar results were obtained for the incorporation by T7 DNA polymerase deficient in 3'-5' exonuclease [T7(exo-)]. To quantify the substitution efficiency, kinetic parameters for incorporation of dOTP and dXTP opposite template C or T by Pol I Kf (exo-) were determined and compared with those for dGTP using oligodeoxynucleotide templates. Incorporation efficiencies (f = Vmax/Km) of dOTP (f = 0.28% min-1 microM-1) and dXTP (f = 0.10% min-1 microM-1) opposite template C were much lower than that of dGTP (f = 1506% min-1 microM-1). Frequencies of mutagenic incorporation of dOTP opposite template T were dependent on the nearest neighbor base pairs, and 1.6-3.9-fold higher than those for dGTP with the nearest neighbors containing G.C pairs. dXTP was not incorporated opposite template T with all four nearest neighbors. These data suggest that formation of dOTP, but not dXTP, from dGTP with nitrous acid or nitric oxide in the intracellular nucleotide pool would result in the elevation of the mutation frequency.