TY - JOUR
T1 - Cupric ion/ascorbate/hydrogen peroxide-induced DNA damage
T2 - DNA-bound copper ion primarily induces base modifications
AU - Drouin, Regen
AU - Rodriguez, Henry
AU - Gao, Shu Wei
AU - Gebreyes, Zewdu
AU - O'Connor, Timothy R.
AU - Holmquist, Gerald P.
AU - Akman, Steven A.
N1 - Funding Information:
Acknowledgements- The authors thank Dr. James Doroshow for helpful discussions, Drs. Richard Cunningham and Serge Boiteux for supplying Nth and Fpg proteins, and Dr. Tim Synold for help with computer simulation. This work was supported by Public Health Service Grant CA53115 from the National Cancer Institute to SAA. R.D. holds a Centennial fellowship from the Medical Research Council of Canada.
PY - 1996
Y1 - 1996
N2 - The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purified human genomic DNA in vitro. Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis. Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which 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 published rule constants. The computer simulation quantitatively predicted: (1) the rate of H2O2 degradation, which was measured using an H2O2-sensitive electrode, (2) the linearity of accumulation of DNA strand breaks and modified bases over the reaction period, (3) the rate of modified base accumulation, and (4) the dependence of modified base and frank strand break production on initial Cu(II) concentration. The simulation significantly overestimated the rate of frank strand break accumulation, suggesting either that the ultimate oxidizing species that attacks the sugar-phosphate backbone is a less-reactive species than the hydroxyl radical used in the model and/or an unidentified hydroxyl radical-scavenging species was present in the reactions. Our experimental data 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.
AB - The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purified human genomic DNA in vitro. Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis. Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which 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 published rule constants. The computer simulation quantitatively predicted: (1) the rate of H2O2 degradation, which was measured using an H2O2-sensitive electrode, (2) the linearity of accumulation of DNA strand breaks and modified bases over the reaction period, (3) the rate of modified base accumulation, and (4) the dependence of modified base and frank strand break production on initial Cu(II) concentration. The simulation significantly overestimated the rate of frank strand break accumulation, suggesting either that the ultimate oxidizing species that attacks the sugar-phosphate backbone is a less-reactive species than the hydroxyl radical used in the model and/or an unidentified hydroxyl radical-scavenging species was present in the reactions. Our experimental data 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.
KW - DNA strand breaks
KW - Free radicals
KW - Oxidized DNA bases
KW - Reactive oxygen species
KW - Transition metals
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U2 - 10.1016/0891-5849(96)00037-8
DO - 10.1016/0891-5849(96)00037-8
M3 - Article
C2 - 8855437
AN - SCOPUS:0029764611
SN - 0891-5849
VL - 21
SP - 261
EP - 273
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
IS - 3
ER -