THE EFFECT OF BLEOMYCIN ON SV40 DNA: CHARACTERISTICS OF BLEOMYCIN ACTION WHICH PRODUCES A SINGLE SCISSION IN A SUPERHELICAL FORM OF SV40 DNA

Abstract

The effect of bleomycin on the molecular integrity of SV40 DNA was determined under various conditions by taking advantage of the fact that a single scission produced in such a superhelical form of DNA could be clearly manifested by analysis of samples with centrifugation in alkaline sucrose gradients. Conditions were found where the reaction velocity, the rate of disappearance of the characteristic form of DNA, depended on the concentration of bleomycin as well as on the reaction time. In contrast to general enzymatic reactions, bleomycin activity was greater at 0°C than at 37°C. The optimum pH was 9.1 while no activity was observed below pH 6 or above pH 13. Additional DNA, irrespective of its source, inhibited the reaction, while RNA had no effect. Cu++ ion at 1 mM almost completely inhibited the reaction while 2-mercaptoethanol at 1 mM stimulated the reaction by about 20-fold. No sulfhydryl compound was included in our standard assay system. EDTA at 10 mM showed no effect.