Degree of DNA unwinding caused by the binding of aclacinomycin A.

The effect of drug binding on the geometry of DNA duplex (plasmid pBR322) has been examined using topoisomerase I relaxation followed by gel electrophoresis. The binding of one molecule of aclacinomycin A was found 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 degrees. To determine the unwinding angle, precise determination of the equilibrium constant of drug-DNA binding-dissociation reaction in the same buffer as that for the topoisomerase reaction (at 37 degrees C) was 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 the 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 binding (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 basis of these experimental results, the possible role of the sugar moieties of these antitumour drugs, as well as that of intercalating chromophores, was discussed.