Copper(II) Facilitates Bleomycin-Mediated Unwinding of Plasmid DNA

The unwinding of plasmid DNA by bleomycin A₂(BLM A₂) was investigated by use of two-dimensional gel electrophoresis. It was found that Cu²⁺ions greatly facilitated the unwinding of topoisomers of plasmid DNA by BLM A₂at concentrations where cupric ions alone had no effect on DNA supercoiling. The concentration of BLM A₂required for observable unwinding was reduced at least 100-fold in the presence of equimolar Cu²⁺. A plot of [Cu²⁺] vs extent of DNA unwinding in the presence of 10^-4M BLM A₂gave a curve consistent with the action of cupric ions on BLM in an allosteric fashion, possibly rearranging the drug into a conformation that facilitates DNA unwinding. The participation of the metal center in enhancing DNA unwinding via direct ionic interaction with one or more negatively charged groups on the DNA duplex also seems possible. Further analysis of the structural factors required for BLM-mediated DNA unwinding was carried out with Cu²⁺+ BLM demethyl A₂, the latter of which differs from BLM A₂only in that it lacks a methyl group, and associated positive charge, at the C-terminus. Cu(II).BLM demethyl A₂was found to be much less effective than Cu(II)-BLM A₂as a DNA unwinding agent, emphasizing the strong dependence of this process on the presence of positively charged groups within the BLM molecule. These findings constitute the first direct evidence that the metal center of BLM can participate in DNA interaction, as well as in the previously recognized role of oxygen binding and activation.