The average unwinding angle of DNA duplex produced by the binding of chromomycin A3

The effect of chromomycin A3 binding on the geometry of DNA duplex (plasmid pBR322) has been examined using topoisomerase I relaxation followed by gel electrophoresis. To determine the equilibrium constant of this drug-DNA binding-dissociation reaction in the same concentration range (ca. 10(-5) M) in the same buffer as those for the topoisomerase reaction (at 37 degrees C), fluorescence measurements were made of the same plasmid-drug system, followed by a Scatchard plot and an analysis using McGhee-von Hippel's exclusion site model. The binding constant has been found to be 3.8 x 10(5) M-1 in the particular buffer (buffer-T) at 37 degrees C, and the number of base pairs involved in the site of one chromomycin molecule on the duplex has been found to be 5. It has been concluded that one chromomycin molecule, bound to the duplex, unwinds it by 11.8 +/- 1.1 degrees. In addition, the enthalpy of binding was determined to be 31.81 kJ/mole using a titration calorimeter with a more concentrated (6.2 mM) solution.