THE AVERAGE UNWINDING ANGLE OF DNA DUPLEX PRODUCED BY THE BINDING OF CHROMOMYCIN A(3)

The effect of chromomycin A, binding on the geometry of DNA duplex (pl asmid pBR322) has been examined using topoisomerase I relaxation follo wed by gel electrophoresis, To determine the equilibrium constant of t his drug-DNA binding-dissociation reaction in the same concentration r ange (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 analysi s 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 bindin g was determined to be 31.81 kJ/mole using a titration calorimeter wit h a more concentrated (6.2 mM) solution.