metallothionein and bathocuproine

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.

Topics: Albumins; Amino Acid Sequence; Animals; Catalase; Copper; DNA; Hydrolysis; Indicators and Reagents; Ligands; Metallothionein; Molecular Sequence Data; Oxidation-Reduction; Phenanthrolines; Rabbits