Ferric and Cupric Ions Requirement for DNA Single-Strand Breakage by H2O2

Abstract

Hydrogen peroxide (H₂O₂), was able to nick the replicative form of the phage fd, without the addition of a reducing agent or of a metal. This DNA single-strand breakage decreased with an increase of the ionic strength, suggesting that H₂O₂ reacted with traces of metal bound to DNA. When cupric of ferric ions were added, the rate of DNA single-strand breakage by H₂O₂ greatly increased and it was 20–30 times faster with cupric than with ferric ions. The addition of EDTA at an equimolar ratio or in excess of metal prevented partially DNA single-strand cleavage by H₂O₂ in the presence of ferric ions and completely when cupric ions were used. Superoxide dismutase prevented DNA single-strand breakage by H₂O₂ and ferric ions. On the contrary, with cupric ions and H₂O₂, the addition of superoxide dismutase increased the rate of DNA single-strand breakage. That superoxide dismutase was acting catalytically was shown by the loss of its effects after heat inactivation of the enzyme. The results of the present study show that besides its involvement in the Fenton reaction, H₂O₂ is able to reduce the metal bound to DNA, generating the superoxide anion radical or/and its protonated form, the perhydroxyl radical involved in DNA nicking. On the other hand, the ability of cuprous ions unlike ferrous ions to dismutate the superoxide radical may explain some differences observed between iron and copper in the DNA single-strand breakage by H,O₂.