Miscoding events occurred at 8-nitro-2’-deoxyguanosine lesion during DNA synthesis by mammalian DNA polymerases

3061

8-nitro-2’-deoxyguanosine (8-NO₂-dG) DNA adducts are endogenously and exogenously induced by the reactive nitrogen species (NO) and may be associated with the development of cancer in inflammatory tissues. To explore the miscoding potential of 8-NO₂-dG adduct, an oligodeoxynucleotide containing a single 8-NO₂-dG adduct was prepared by photochemical synthesis and used as a template in primer extension reactions catalyzed by mammalian DNA polymerases. Primer extension reactions catalyzed by pol α or β were retarded at the 8-NO₂-dG lesion; a fraction of primers was extended past the lesion. The fully extended products were analyzed using a two-phased polyacrylamide gel electrophoresis to quantify the miscoding frequency and specificity occurred at the lesion site. Both the enzymes promoted preferential incorporation of dCMP, the correct base, opposite the 8-NO₂-dG lesion, accompanied by lesser amounts of misincorporation of dAMP; the frequency of base substitutions induced by pol α was much higher than that of pol β. Pol η and κ showed more broad miscoding spectra; direct incorporations of dCMP and damp were observed, along with lesser amounts of dGMP and dTMP incorporations and deletions. The miscoding frequencies observed with pol η and κ were much higher than that of pol α or β. These observations were supported by steady-state kinetic studies. 8-NO₂-dG is a miscoding lesion, generating primarily G → T transversions in mammalian cells. Miscoding frequency and specificity of 8-NO₂-dG varied depending on DNA polymerase used.