OXYGEN RADICAL-INDUCED SINGLE-STRAND DNA BREAKS AND REPAIR OF THE DAMAGE IN A CELL-FREE SYSTEM

Ferric nitrilotriacetate (Fe3+-NTA) catalyzes hydrogen peroxide-derive d production of hydroxyl radicals, which are known to cause DNA damage . In the present work, Fe3+-NTA plus hydrogen peroxide-induced single- strand DNA breaks and repair of the DNA damage were studied in vitro b y monitoring DNA damage- and DNA repair-dependent conformational chang es of pUC18 plasmid DNA. Single-strand DNA breaks were induced in the pUC18 DNA by Fe3+-NTA plus hydrogen peroxide in a dose-dependent fashi on. Induction of the DNA damage was inhibited by deferoxamine mesylate (an iron chelator) and by hydroxyl radical scavengers such as dimethy l sulfoxide (DMSO), D-mannitol and ethanol indicating that the DNA dam age was caused by hydroxyl radicals which were generated by reaction o f Fe3+-NTA with hydrogen peroxide. The oxygen radical-induced single-s trand DNA breaks were repaired partly (more than 50%) by incubating th e damaged DNA at 37 degrees C for 3 h with a partially purified prepar ation of APEX nuclease (a multifunctional DNA repair enzyme), DNA poly merase beta, four deoxyribonucleoside triphosphates, T4 DNA ligase and ATP. Analyses of the partially purified preparation of APEX nuclease revealed that a 45-kDa protein as well as APEX nuclease in the prepara tion were involved in the repair of the single-strand DNA breaks. APEX nuclease was suggested to initiate the repair by removing 3' termini blocked by the nucleotide fragments and also by incising the 5' side o f AP sites. The 45-kDa protein was suggested to be required for remova l of the 5' tags such as 5'-terminal deoxyribose phosphate residues pr oduced by the action of APEX nuclease on AP sites.