Molecular Aspects of Bleomycin-Promoted Damage of Covalently Closed Circular DNA

An extensive amount of information has been accumulated concerning the extracellular degradation of DNA by bleomycin since its discovery in 1966 by Umezawa et al. (1). It has been established that the drug causes the release of free bases, ruptures the phosphodiester backbone, and damages the deoxyribose moiety of DNA. There are no detectable damaging effects on RNA, nor does the drug alter the ability of mRNA to direct cell-free protein synthesis. For a complete review of both the intra- and extracellular effects of bleomycin, the reader is referred to several recent review articles(2-4). Most of the previous studies on the extracellular DNA fragmentation reaction have relied heavily on the use of velocity sedimentation analyses (usually sucrose gradients) to assay for the introduction of strand scissions into DNA. Additionally, most of these studies employed linear duplex DNA as the substrate for the reaction. The use of linear DNA in conjunction with velocity sedimentation analyses requires the introduction of many single- or double-strand breaks into the DNA before an appreciable (and measurable) decrease in the sedimentation velocity occurs. The use of such a limited technology precludes the possibility of asking some of the more fundamental questions concerning molecular mechanisms which are operative in this bleomycin-DNA reaction at low drug concentrations. To answer some of these basic questions requires the use of a defined DNA substrate that is homogeneous with respect to size and base sequence content.