Use of repair endonucleases to characterize DNA damage induced by reactive oxygen species in cellular and cell-free systems

Abstract

A number of repair endonucleases, viz. endonuclease III, formamidopyrimidine-DNA glycosylase (FPG protein), endonuclease IV, exonuclease III and UV endonuclease, is used to simultaneously quantify various types of DNA modifications, which were induced by agents that generate reactive oxygen species. Under cell-free conditions, two types of DNA damage profiles are obtained. The profiles induced by chemically generated singlet oxygen and by various photosensitizers (acridine orange, methylene blue, riboflavin, hematoporphyrin) plus light are dominated by base modifications sensitive to FPG protein, while 5,6-dihydropyrimidines (recognized by endonuclease III), sites of base loss (AP sites, recognized by endonuclease IV and exonuclease III) and strand breaks are minor lesions. In contrast, the DNA damage profile induced by hydroxyl radicals (γ-rays) consists of approx. equal levels of base modifications, AP sites and strand breaks. The damage profiles induced by Fe(III)-EDTA in the presence of Superoxide and by Fe(III)-nitrilotriacetate in the presence of H 2O 2 do not differ from that by hydroxyl radicals. The damage profile induced by Cu(II)-phenanthroline deviates by high levels of AP sites that are recognized by endonuclease IV and exonuclease III — but not by those AP endonucleases which cleave at the 3' site — and probably represent AP sites oxidized at C-1'. The damage induced by Fe(III)-bleomycin plus H 2O 2 deviates by an increased level of double strand breaks and the absence of endonuclease-sensitive base modifications. Cellular DNA damage profiles are obtained from bacteria, cultured mammalian cells and mammalian mitochondria after exposure to acridine orange plus visible light. A comparison with the cell-free profiles reveals that the damage in all three systems is not induced indirectly by hydroxyl radicals or an activation of cellular nueleases, but by the same mechanism that is responsible for the cell-free DNA damage