DEGREE OF DNA UNWINDING CAUSED BY THE BINDING OF ACLACINOMYCIN-A

The effect of drug binding on the geometry of DNA duplex (plasmid pBR3 22) has been examined using topoisomerase I relaxation followed by gel electrophoresis, The binding of one molecule of aclacinomycin A was f ound to cause an unwinding of the DNA double helix by an angle of 8+/- 2 degrees in aqueous solution at 37 degrees C, The unwinding angle of daunomycin was 12+/-2 degrees, and that of ethidium bromide 15+/-3 deg rees, To determine the unwinding angle, precise determination of the e quilibrium constant of drug-DNA binding-dissociation reaction in the s ame buffer as that for the topoisomerase reaction (at 37 degrees C) wa s indispensable, This determination was made by ultraviolet absorption measurement of the same plasmid-drug system, followed by a Scatchard plot and analysis using McGhee-von Hippel's excluded site model, For t he aclacinomycin-pBR322 system, the binding constant (K) was 7.2 x 10( 4) M-1, and the number of base pairs in the single site of drug bindin g (n) was 4.0, For daunomycin-pBR322, K = 7.1 x 10(4) M-1 and n = 3.4, and for ethidium-pBR322, K = 4.0 x 10(4) M-1 and n = 3.3, On the basi s of these experimental results, the possible role of the sugar moieti es of these antitumour drugs, as well as that of intercalating chromop hores, was discussed.