CUPRIC ION ASCORBATE HYDROGEN PEROXIDE-INDUCED DNA-DAMAGE - DNA-BOUNDCOPPER-ION PRIMARILY INDUCES BASE MODIFICATIONS

The kinetics of frank DNA strand breaks and DNA base modifications pro duced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purif ied human genomic DNA in vitro. Modified bases were determined by clea vage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency b efore (frank strand breaks) and after (modified bases) Nth or Fpg prot ein digestion was quantified by neutral glyoxal gel electrophoresis. D ialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weig ht species, probably transition metal ions and metal ion chelators, wh ich supported frank strand breaks in the presence of ascorbate + H2O2 without supplemental copper ions. We then established a kinetic model of the DNA-damaging reactions caused by Cu(II) + ascorbate + H2O2 The principal new assumption in our model was that DNA base modifications were caused exclusively by DNA-bound Cu(I) and frank strand breaks by non-DNA-bound Cu(I). The model was simulated by computer using publish ed rate constants. The computer simulation quantitatively predicted: ( 1) the rate of H2O2 degradation, which was measured using an H2O2-sens itive electrode, (2) the linearity of accumulation of DNA strand break s and modified bases over the reaction period, (3) the rate of modifie d base accumulation, and (4) the dependence of modified base and frank strand break production on initial Cu(II) concentration. The simulati on significantly overestimated the rate of frank strand break accumula tion, suggesting either that the ultimate oxidizing species that attac ks the sugar-phosphate backbone is a less-reactive species than the hy droxyl radical used in the model and/or an unidentified hydroxyl radic al-scavening species was present in the reactions. Our experimental da ta are consistent with a model of copper ion-DNA interaction in which DNA-bound Cu(I) primarily mediates DNA base modifications and nonbound Cu(I) primarily mediates frank strand break production.