Binding of mithramycin to DNA in the presence of second drugs

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Abstract
Comparative DNA equilibrium binding studies with mithramycin (MTR) and ethidium bromide in the presence and in the absence of second drugs were investigated by spectral titrations. Unusual curvatures (in contrast to those due to neighbor exclusion or anticooperativity) are found in the Scatchard plots of MTR-DNA titrations in the presence of netropsin, a minor-groove binder. Parallel studies with ethidium bromide indicate that although the presence of netropsin significantly reduces the binding ability of ethidium no unusually curved Scatchard plots are obtained. The unusual curvature exhibited by the Scatchard plots of MTR titrations in the presence of netropsin indicates that the binding of netropsin greatly affects the MTR binding to DNA and can be simulated by an explicit incorporation of the second drug-DNA interaction in the binding formalism. Since netropsin is a minor-groove binder, its interference with the binding of MTR is in accord with the notion that MTR also binds at this groove. The observation of negligible effects on the DNA binding ability of MTR in the presence of either a major-groove or a phosphate group binder lends further support to this conclusion. Consistent with its guanine specificity, studies with synthetic polynucleotides suggest that MTR exhibits negligible affinity for poly(dA-dT) .cntdot. poly(dA-dT) or poly-(dA) .cntdot. poly(dT). Although the strongest bindings are demonstrated by poly(dG-m5dC) .cntdot. poly(dG-m5dC), poly(dG-dC) .cntdot. poly(dG-dC), and poly (dG) .cntdot. poly (dC), the homopolymer exhibits a saturation density of approximately 1 drug molecule per every 2-3 base pairs, whereas the alternating polymers saturate at 1 drug molecule per < 2 base pairs, a rather surprising finding for such an extended molecule but one consistent with the drug-dimer model on binding. The effect of sequence or conformation on the binding is further indicated by the stronger MTR affinity for poly(dA-dG) .cntdot. poly(dC-dT) than for poly(dA-dC) .cntdot. poly(dG-dT). These results are supported by circular dichroic measurements.