DISTINCT MECHANISMS OF SITE-SPECIFIC DNA-DAMAGE INDUCED BY ENDOGENOUSREDUCTANTS IN THE PRESENCE OF IRON(III) AND COPPER(II)

The ability of Cu(II) and Fe(III) to promote site-specific DNA damage in the presence of endogenous reductants was investigated by using P-3 2-5'-end-labeled DNA fragments obtained from the human p53 tumor suppr essor gene and the c-Ha-ras-1 protooncogene. Ascorbate induced metal-d ependent DNA damage most efficiently (ascorbate > GSH > NADH). Cu(II) induced endogenous reductants-dependent DNA damage more efficiently th an Fe(III). Endogenous reductants plus Fe(III) caused DNA cleavage at every nucleotide, without marked site preference. DNA damage by Fe(III ) was inhibited by hydroxyl free radical ((OH)-O-.) scavengers and cat alase. These results suggest that endogenous reductants plus Fe(III) g enerate free or extremely near free (OH)-O-. via H2O2 formation, and t hat (OH)-O-. causes DNA damage. In the presence of 50 mu M Cu(II) in b icarbonate buffer, ascorbate caused DNA cleavage frequently at sites o f two or more adjacent guanine residues. In contrast, in the presence of 20 mu M Cu(II), ascorbate caused DNA cleavage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited DNA damage by Cu(II), whereas (OH)-O-. scavengers did not. Fe(III)-dependent 8-o xo-7,8-dihydro-2'-deoxyguanosine formation was inhibited by (OH)-O-. s cavengers, whereas no inhibition by (OH)-O-. scavengers was observed w ith Cu(II). These results suggest that (OH)-O-. is the main active spe cies formed with Fe(III), whereas copper-peroxide complexes with a rea ctivity similar to (OH)-O-. participate in Cu(II)-dependent DNA damage . The polyguanosine sequence specificity of DNA damage in the presence of high concentrations of Cu(II) can be explained by the preferential binding of Cu(II) to guanine residues. (C) 1998 Elsevier Science B.V. All rights reserved.