MISINCORPORATION OF 2'-DEOXYOXANOSINE 5'-TRIPHOSPHATE BY DNA-POLYMERASES AND ITS IMPLICATION FOR MUTAGENESIS

2'-Deoxyoxanosine (dOxo) is a novel DNA lesion produced by the reactio n of 2'-deoxyguanosine (dGuo) with nitrous acid and nitric oxide [Suzu ki, T., Yamaoka, R., Nishi, M., Ide, H., and R Makino, K. (1996) J. Am . Chem. Sec. 118, 2515-2516]. In this work, 2'-deoxyoxanosine 5'-triph osphate (dOTP) was prepared by nitrous acid treatment of 2'-deoxyguano sine 5'-triphosphate (dGTP), and its incorporation into DNA by DNA pol ymerases was investigated to elucidate the substrate and mutagenic pro perties of dOTP. Primed M13mp18 DNA was replicated by Escherichia coli DNA polymerase I Klenow fragment (Pal I Kf) in the presence of three normal dNTPs and dOTP or 2'-deoxyxanthosine 5'-triphosphate (dXTP), an other major product of reaction of dGTP with nitrous acid and nitric o xide. 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' exonucl ease [T7(exo(-))]. To quantify the substitution efficiency? kinetic pa rameters for incorporation of dOTP and dXTP opposite template C or T b y Pol I Kf (exo-) were determined and compared with those for dGTP usi ng oligodeoxynucleotide templates. Incorporation efficiencies (f = V-m ax/K-m) of dOTP (f = 0.28% min(-1) mu M-1) and dXTP (f = 0.10% min(-1) mu M-1) opposite template C were much lower than that of dGTP (f = 15 06% min(-1) mu M-1), Frequencies of mutagenic incorporation of dOTP op posite template T were dependent on the nearest neighbor base pairs, a nd 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 wi th all four nearest neighbors. These data suggest that formation of dO TP, but not dXTP, from dGTP with nitrous acid or nitric oxide in the i ntracellular nucleotide pool would result in the elevation of the muta tion frequency.