Oxidative and nonoxidative mechanisms of site-specific DNA cleavage induced by copper-containing metallothioneins.
DNA cleavage induced by metallothionein (MT) containing copper was investigated by a DNA sequencing technique. Reconstituted Cd7-MT showed no ability to cause DNA cleavage. Commercially available rabbit MT I caused DNA cleavage, suggesting that DNA cleavage is due to the metal contained in commercial Mt. Cu2Cd5-MT and Cu12-MT were prepared by the treatment of commercial rabbit MT I with [Cu(CH3CN)4]CIO4. Cu12-MT frequently induced an alteration of thymine residues, especially in the 5'-GTC-3' sequence, and piperidine treatment led to chain cleavage at the thymine residues. The site specificity was similar to that obtained with Cu(I) plus H2O2. H2O2 enhanced DNA cleavage induced by Cu12-MT. Catalase and a Cu(I)-specific chelating agent, bathocuproine, inhibited DNA cleavage. These results suggest that Cu(I) and H2O2 have important roles in the production of active species causing DNA cleavage. Commercial MT and Cu2Cd5-MT induced DNA cleavage much less than Cu12-MT, but gave particularly specific DNA cleavage. Cu2Cd5-MT induced cleavage specifically at the central guanine residue of the 5'-GGT-3' sequence. A similar cleavage pattern was obtained with commercial MT. No effect of piperidine treatment suggests that the DNA cleavage might not be due to base damage and/or liberation. The DNA cleavage was inhibited efficiently by EDTA, but not by bathocuproine and catalase. Experiments with DNA ligands, albumin, and denatured DNA suggest that commercial MT and Cu2Cd5-MT induce nonoxidative cleavage of the deoxyribose phosphate backbone through its DNA recognition. These two types of cleavage mechanisms are discussed in relation to the possible role of Cu-MT in carcinogenesis.
Oikawa
S
S
Department of Public Health, Faculty of Medicine, Kyoto University, Japan.
Kurasaki
M
M
Kojima
Y
Y
Kawanishi
S
S
D000418:Albumins / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002374:Catalase / Q000737:chemistry; D003300:Copper / Q000032:analysis*; D004247:DNA / Q000378:metabolism*; D006868:Hydrolysis; D007202:Indicators and Reagents; D008024:Ligands; D008668:Metallothionein / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000737:chemistry; D011817:Rabbits
D000418:Albumins; D007202:Indicators and Reagents; D008024:Ligands; D010618:Phenanthrolines; D003300:Copper; D004247:DNA; D008668:Metallothionein; C002478:bathocuproine; D002374:Catalase