Bleomycin-induced Alkaline-labile Damage and Direct Strand Breakage of PM2 DNA

Bleomycin-induced breakage of an isolated covalently closed circular DNA from bacteriophage PM2 was assayed fluorometrically after agarose gel electrophoresis and staining with ethidium bromide. When bleomycin-damaged DNA was assayed under neutral conditions, there was a decrease in the amount of Form I DNA and a simultaneous increase in both Forms II and III of DNA. However, when the damage was assayed under nondenaturing alkaline conditions, there was a greater decrease in the amount of Form I DNA and a corresponding increase in both Forms II and III DNA compared with neutral conditions. Approximately one alkaline-labile site was formed for every single-strand break introduced. The rate of formation of Form III DNA was found to be approximately twice as fast when measured under alkaline conditions compared with neutral condftions. Reaction of bleomycin-treated PM2 DNA with the Escherichia coil apurinic specific endonuclease IV demonstrated that the loss of Form I DNA was very similar to that observed with alkali treatment suggesting that the bleomycin-induced alkaline-labile damage is the result of base removal from the DNA helix. Similar results were obtained when the bleomycin-treated DNA was reacted with E. coli endonuclease III. However, no increase in double-strand breakage was observed with either endonuclease III or IV, which may reflect an inability of the endonuclease to cleave at a site of base loss across from a single-strand break or another lost base. Alkali treatment of purified Form I DNA which was pretreated with bleomycin revealed that some of the alkali-induced double-strand breaks arose from Form I molecules, suggesting that two alkaline-labile sites are formed across from each other. In addition, some Form II molecules produced by bleomycin treatment were converted to Form III molecules by non-denaturing alkaline conditions.