How and How Much Can Hoechst 33258 Cause Unwinding in a DNA Duplex?

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Abstract
The effect of Hoechst 33258 binding on the geometry of a DNA duplex (plasmid pBR322) has been examined using topoisomerase II relaxation followed by gel electrophoresis. Of this drug-DNA system, fluorescence, optical absorption, and calorimetric measurements were also made at various drug and DNA concentrations and in the same buffer as that for the topoisomerase reaction. It has been confirmed that there are two modes of drug-DNA interaction. When the drug concentration is much lower than the DNA base pair concentration, the Hoechst 33258 molecule binds in the minor groove of the DNA duplex and occupies a site formed of five continuous base pair sequences that contain no G·C pair. Here, the equilibrium constant K1 is 1.8×107M-1 (at 37°C), and the enthalpy of binding ΔH1 is -865 cal/mol. When the drug concentration is much higher, on the other hand, it shows another binding mode which is much weaker, so that K2=2.25×104M-1 and ΔH2 is -464 cal/mol, which gives fluorescence quenching, which has no base pair preference, and which causes an unwinding of the duplex by 1 degree.

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