Copper-dependent cleavage of DNA by bleomycin

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Abstract
DNA strand scission by bleomycin in the presence of Cu and Fe was further characterized. It was found that DNA degradation occurred readily upon admixure of Cu9I) or Cu(II) + dithiothreitol + bleomycin, but only where the order of addition precluded initial formation of Cu(II)-bleomycin or where sufficient time was permitted for reduction of the formed Cu(II)-bleomycin to Cu(I)-bleomycin. DNA strand scission mediated by Cu + dithiothreitol + bleomycin was inhibited by the copper-selective agent bathocuprione when the experment was carried out under conditions consistent with Cu chelation by bathocuprione on the time scale of the experiment. Remarkably, it was found that the extent of DNA degrdation obtained with bleomycin in the presence of Fe and Cuwas greater than that obtained with either metal ion alone. A comparison of the sequence selectivity of bleomycin in the presence of Cu and Fe using 32P-end-labeled DNA duplexes as substrates revealed significant differences in site sof DNA cleavage and in the extent of cleavage at sites shared in common. For deglycobleomycin and decarbamoylbleomycin, whose metal ligation is believed to differ from that of bleomycin itelf, it was found that the relative extents of DNA cleavage in the presence of Cu were not in the same order as those obtained in the presence of Fe. The bleomycin-mediated oxygenation products derived from cis-stilbene were found to differ in type and amount in the presence of added Cu vs. added Fe. Interestingly, while product formation from cis-stilbene was decreased when excess Fe was added to a reaction mixture containing 1:1 Fe(III) and bleomycin, the extent of product formation was enhanced almost 4-fold in reactions that contained 5:1, as compared to 1:1, Cu and bleomycin. The results of these experiments are entirely consistent with the work of Sugiura [Sugiura, y. (1979) Biochem. Biophys. Res. Commun. 90, 375-383], who first demonstrated the generation of reactive oxygen species upon admixture of O2 and Cu(I)-bleomycin.