Oxidative damage to DNA, reflected in the formation of 8-oxo-7-hydrodeoxyguanosine (8-oxodG), may be important in mutagenesis, carcinogenesis and the ageing process. Kuchino et al. studied DNA synthesis on oligodeoxynucleotide templates containing 8-oxodG, concluding that the modified base lacked base pairing specificity and directed misreading of pyrimidine residues neighbouring the lesion. Here we report different results, using an approach in which the several products of a DNA polymerase reaction can be measured. In contrast to the earlier report, we find that dCMP and dAMP are incorporated selectively opposite 8-oxodG with transient inhibition of chain extension occurring 3' to the modified base. The potentially mutagenic insertion of dAMP is targeted exclusively to the site of the lesion. The ratio of dCMP to dAMP incorporated varies, depending on the DNA polymerase involved. Chain extension from the dA.8-oxodG pair was efficiently catalysed by all polymerases tested.

Endemic (Balkan) nephropathy (EN),environmental mutagen ͉ p53 mutation ͉ urothelial cancer ͉ DNA adduct

Substrate specificities of FPG protein (also known as formamidopyrimidine DNA glycosylase) and 8-hydroxyguanine endonuclease were compared by using defined duplex oligodeoxynucleotides containing single residues of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA), and 2,6-diamino-4-hydroxy-5-(N-methyl)formamidopyrimidine (Me-Fapy). Duplexes containing 8-oxodG positioned opposite dC, dG, or dT were cleaved, whereas single-stranded DNA and duplexes containing 8-oxodGdA or 8-oxodA positioned opposite any of the four DNA bases were relatively resistant. Both enzymes cut duplexes containing 8-oxoG-dC 3' and 5' to the modified base but failed to cleave duplex DNA containing synthetic abasic sites, mismatches containing dG, or unmodified DNA. 8-Oxoguanine, identified by HPLC-electrochemical detection techniques, was released during the enzymatic reaction. Apparent Km values for FPG protein acting on duplex substrates containing a single Me-Fapy or 8-oxodG residue positioned opposite dC were 41 and 8 nM, respectively, and those for 8-hydroxyguanine endonuclease were 30 and 13 nM, respectively. Comparison of the properties of the two enzyme activities suggest that they are identical. In view of the widespread distribution of 8-oxodG in cellular DNA, the demonstrated miscoding and mutagenic properties of this lesion, and the existence of a bacterial gene coding for FPG protein, we propose that 8-oxodG DNA is the primary physiological substrate for a constituent glycosylase found in bacteria and mammalian cells.Active oxygen species, generated by ionizing radiation and by endogenous oxidation processes, react with deoxyguanosine (dG) residues in DNA to form 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) (reviewed in ref.

Proton NMR studies are reported on the complementary d(C1-C2-A3-C4-T5-A6-oxo-G7-T8-C9-A10-C11-C12).d(G13-G14-T15- G16-A17-A18-T19- A20-G21-T22-G23-G24) dodecanucleotide duplex (designated 8-oxo-7H-dG.dA 12-mer), which contains a centrally located 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) residue, a group commonly found in DNA that has been exposed to ionizing radiation or oxidizing free radicals. From the NMR spectra it can be deduced that this moiety exists as two tautomers, or gives rise to two DNA conformations, that are in equilibrium and that exchange slowly. The present study focuses on the major component of the equilibrium that originates in the 6,8-dioxo tautomer of 8-oxo-7H-dG. We have assigned the exchangeable NH1, NH7, and NH2-2 base protons located on the Watson-Crick and Hoogsteen edges of 8-oxo-7H-dG7 in the 8-oxo-7H-dG.dA 12-mer duplex, using an analysis of one- and two-dimensional nuclear Overhauser enhancement (NOE) data in H2O solution. The observed NOEs derived from the NH7 proton of 8-oxo-7H-dG7 to the H2 and NH2-6 protons of dA18 establish an 8-oxo-7H-dG7(syn).dA 18(anti) alignment at the lesion site in the 8-oxo-7H-dG.dA 12-mer duplex in solution. This alignment, which places the 8-oxo group in the minor groove, was further characterized by an analysis of the NOESY spectrum of the 8-oxo-7H-dG.dA 12-mer duplex in D2O solution. We were able to detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(A6-oxo-G7-T8).d(A17-A18-T19) trinucleotide segment centered about the lesion site that establishes stacking of the oxo-dG7(syn).dA(anti) pair between stable Watson-Crick dA6.dT19 and dT8.dA17 base pairs with minimal perturbation of the helix. Thus, both strands of the 8-oxo-7H-dG.dA 12-mer duplex adopt right-handed conformations at and adjacent to the lesion site, the unmodified bases adopt anti glycosidic torsion angles, and the bases are stacked into the helix. The energy-minimized conformation of the central d(A6-oxo-G7-T8).d(A17-A18-T19) segment requires that the 8-oxo-7H-dG7(syn).dA18(anti) alignment be stabilized by two hydrogen bonds from NH7 and O6 of 8-oxo-7H-dG7(syn) to N1 and NH2-6 of dA18(anti), respectively, at the lesion site.(ABSTRACT TRUNCATED AT 400 WORDS)