HO* radicals induce an unexpected high proportion of tandem base lesions refractory to repair by DNA glycosylases

Abstract

Reaction of HO(*) radicals with double-stranded calf thymus DNA produces high levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and, to a minor extent, 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodAdo). Formation of the hydroxylated purine lesions is explained by addition of HO(*) to the C8 position of the purine moiety. It has been reported that tandem lesions containing a formylamine residue neighboring 8-oxodGuo could be produced through addition of a transiently generated pyrimidine peroxyl radical onto the C8 of an adjacent purine base. Formation of such tandem lesions accounted for approximately 10% of the total 8-oxodGuo. In the present work we show that addition of HO(*) onto the C8 of purine accounts for only approximately 5% of the generated 8-oxodGuo. About 50% of the 8-hydroxylated purine lesions, including 8-oxodGuo and 8-oxodAdo, are involved in tandem damage and are produced by peroxyl addition onto the C8 of a vicinal purine base. In addition, the remaining 45% of the 8-oxodGuo are produced by an electron transfer reaction, providing an explanation for the higher yield of formation of 8-oxodGuo compared to 8-oxodAdo. Interestingly, we show that >40% of the 8-oxodGuo involved in tandem lesions is refractory to excision by DNA glycosylases. Altogether our results demonstrate that, subsequently to a single oxidation event, peroxidation reactions significantly increase the yield of formation of hydroxylated purine modifications, generating a high proportion of tandem lesions partly refractory to base excision repair.

Scheme 1.

Proposed mechanisms of HO^•-mediated formation of 8-hydroxylated purine lesions in double-stranded DNA. Path A involves the well-defined mechanism of HO^• addition to the C8 of the purine base producing single lesions. Tandem lesions are produced by addition of a peroxyl radical onto the C8 of an adjacent purine base followed by decomposition of the transiently generated endoperoxide (path B). Electron transfer from mostly guanine to peroxyl radicals (path C) explains the high yield of formation of 8-oxodGuo observed in double-stranded DNA.

Fig. 1

Determination of the origin of the incorporated oxygen atom in 8-oxoPur upon HO^•-mediated DNA, nucleoside, or polynucleotide oxidation. (A) Percentage of incorporation of the ¹⁸O-labeled oxygen atom in ThdGly, 8-oxodGuo, and 8-oxodAdo upon gamma irradiation of an aqueous aerated DNA solution in the presence of either ¹⁸O-labeled water (H₂¹⁸O, 95% enrichment, open bars) or ¹⁸O-labeled molecular oxygen (¹⁸O₂, 76% enrichment, hatched bars). (B) Percentage of generated 8-oxodGuo (shaded bars) and 8-oxodAdo (hatched bars) having incorporated an ¹⁸O atom upon gamma radiolysis of an aerated aqueous H₂¹⁸O (95% enrichment) solution of dAdo, a mixture of nucleosides (dNs), or double-stranded polynucleotides containing specific DNA bases. Results represent the average and standard deviation of four independent determinations.

Fig. 2.

Yields of formation of 8-oxodGuo (shaded bars) and 8-oxodAdo (hatched bars) expressed as the number of generated lesions per million normal DNA bases and per Gy upon gamma radiolysis of aqueous aerated solutions of either DNA or double-stranded polynucleotides containing specific DNA bases. Results represent the average and standard deviation of at least three independent experiments.

Fig. 3.

Remaining amounts of 8-oxodGuo as single damage (open bars) or involved in tandem lesions (shaded bars) upon incubation of gamma-irradiated DNA with hOGG1 at 37 °C for increasing periods of time. Results represent the average and standard deviation of three independent determinations.