Bleomycin-induced alkaline-labile damage and direct strand breakage of PM2 DNA
Lloyd, R.S.; Haidle, C.W.; Hewitt, R.R.
Cancer Research 38(10): 3191-3196
1978
ISSN/ISBN: 0008-5472 PMID: 80262 Document Number: 135742
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 l DNA and a simultaneous increase in Forms ll and lll of DNA. When the damage was assayed under nondenaturing alkaline conditions, there was a greater decrease in the amount of Form l DNA and a corresponding increase in Forms ll and lll DNA compared with neutral conditions. Approximately 1 alkaline-labile site was formed for every single-strand break introduced. The rate of formation of Form lll DNA was approximately twice as fast when measured under alkaline conditions compared with neutral conditions. Reaction of bleomycin-treated PM2 DNA with the Escherichia coli apurinic specific endonuclease lV demonstrated that the loss of Form l 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. No increase in double-strand breakage was observed with 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 2 alkaline-labile sites were formed across from each other. Some Form II molecules produced by bleomycin treatment were converted to Form III molecules by nondenaturing alkaline conditions.