Site-specific DNA damage induced by cobalt(II) ion and hydrogen peroxide: role of singlet oxygen

Abstract

The effect of Co(II) ion on the reaction of hydrogen peroxide with DNA was investigated by a DNA sequencing technique using 32P-5'-end-labeled DNA fragments obtained from human c-Ha-ras-1 protooncogene. Co(II) induced strong DNA cleavage in the presence of hydrogen peroxide even without alkali treatment. Guanine residues were the most alkali-labile site, and the extent of cleavages at the positions of thymine and cytosine was dependent on the sequence. Adenine residues were relatively resistive. Diethylenetriaminepentaacetic acid, present in excess over Co(II), inhibited DNA cleavage. Singlet oxygen scavengers (dimethylfuran, sodium azide, 1,4-diazabicyclo[2.2.2]octane, dGMP), sulfur compounds (methional, methionine), and superoxide dismutase inhibited DNA cleavage completely. Hydroxyl radical scavengers were not so effective as singlet oxygen scavengers. ESR studies using 2,2,6,6-tetramethyl-4-piperidone as a singlet oxygen trap suggest that Co(II) reacts with hydrogen peroxide to produce singlet oxygen or its equivalent. ESR studies using 5,5-dimethylpyrroline N-oxide (DMPO) showed that the hydroxyl radical adduct of DMPO was also formed. The results suggest that Co(II) ion binds to DNA and subsequently reacts with hydrogen peroxide to produce singlet oxygen and hydroxyl radicals and that singlet oxygen plays a more important role in the DNA damage than hydroxyl free radicals.