Modulation of DNA breakage induced via the Fenton reaction

The conversion of the covalently closed circular double-stranded supercoiled DNA (pBR322) to a relaxed circle was used to investigate DNA nicking induced by Fe2+ and H2O2. In our experimental conditions of ionic strength (150 mM NaCl), pH = 7 and temperature (37 degrees C), the dose-response curve for the ferrous iron mediated H2O2 dependent DNA nicking is peculiar. For a fixed concentration of ferrous iron (2 microM), the concentration of H2O2 producing a maximum extent of DNA nicking was about 10-30 microM. The DNA single-strand breakage decreased with an increase of H2O2 concentration. We have investigated the effects of several factors such as the nature of the buffer, ionic strength, temperature and pH. Buffer components leading to the autoxidation of ferrous iron to ferric iron (phosphate) or to the scavenging of reactive oxygen species (Tris) greatly alter the dose-response curve. The H2O2 concentrations required for producing the maximum extent of DNA single-strand breaks at 4 degrees C and 56 degrees C were respectively 30 microM and 3 microM. At pH = 10, the pattern of the dose-response curve was totally different. The data showed that the peculiar dose-response curve for the ferrous iron mediated H2O2 dependent DNA nicking greatly depended on the experimental conditions.