Bleomycin-induced DNA lesions at mutational hot spots: implications for the mechanism of double-strand cleavage.

Abstract

Using various end-labeled, defined-sequence DNA substrates, we examined bleomycin-induced damage at several G'C base pairs which correspond to mutational hot spots. The most frequent lesions detected were single-strand breaks and single apu inic/apyrimidinic (AP) sites at the C residue, suggesting that this was the primary site of damage. Strand breaks and AP sites also occurred, but less frequently, at a secondary damage site-i.e., the directly opposed G residue in the complementary strand. However, damage at the secondary site occurred only when a strand break was present at the primary site, and AP sites at the primary site were never accompanied by closely opposed damage in the complementary strand. Thus, formation of a strand break at the primary damage site was a necessary though not sufficient condition for attack at the secondary site. Similar patterns were seen at other sequences attacked by bleomycin, although primary and secondary sites were sometimes staggered by one nucleotide position rather than directly opposed. These and other results suggest a mechanism of double-strand cleavage in which bleomycin Is reactivated during formation of the first strand break, and the reactivated drug subsequently attacks the complementary strand at a specific position which is not normally a site of bleomycin-induced cleavage. Regeneration of activated bleomycin could result from a reaction between Fe(w)'bleomycin and a 4'-peroxyl derivative of deoxyribose, both produced during formation of the strand break.The fact that bleomycin produces double-strand breaks with single-hit kinetics suggests that these lesions result from a single interaction between bleomycin and DNA (1, 2). However, the chemistry of activated bleomycin (3,4) gives no suggestion of bifunctionality, and the mechanism of doublestrand cleavage has remained uncertain. We recently showed that most bleomycin-induced double-strand breaks consist of a "primary" site, which follows the normal G-Y (Y =pyrim-idine nucleoside) specificity characteristic of bleomycininduced single-strand cleavage, and a secondary site (in the opposite strand), which is seldom a G-X sequence and is usually a site where single-strand cleavage is rare (2). In addition to double-strand breaks, bleomycin induces formation of apurinic/apyrimidinic (AP) sites with closely opposed strand breaks, and these bivalent lesions have been implicated in bleomycin-induced mutagenesis in A phage (5, 6). To clarify the mechanism by which bleomycin effects concomitant damage to both DNA strands, we have examined the detailed structure of bivalent lesions induced by bleomycin at specific sites in defined-sequence DNA substrates, particularly those corresponding to mutational hot spots.MATERIALS AND METHODS Materials. Recombinant plasmids were constructed by cloning either a synthetic 25-mer (pc1245, Fig. 1 Analysis of DNA Cleavage. Nondenaturing and denaturing gel electrophoresis and elution of fragments from gels have been described (2). Except in experiments employing hydrazine, all e...

Chemical and Biological Evidence for Base Propenals as the Major Sourceof the Endogenous M1dG Adduct in CellularDNA

“…However, there is one mechanistic distinction between the two sets of oxidants used in the present studies. Current evidence suggests that DNA-bound bleomycin participates in the subsequent chemistry of the deoxyribose oxidation it initiates (38,39), whereas degradation of ONOO Ϫ simultaneously produces both hydroxyl radical and nitrogen dioxide radical (reviewed in Ref. 40).…”

DNA Cleavage/Repair and Signal Transduction Pathways in Irradiated Breast Tumor Cells

“…We failed to observe any direct evidence of cell killing in MCF-7 cells with either l,25-(OH) 2 -D 3 orEB 1089 with exposure times as long as 96 h, although it is possible that the reduction in the number of cells over time reflects both growth inhibition and (a limited degree of) cell death. While other laboratories have reported apoptosis in breast tumor cells (including MCF-7 cells) treated with vitamin D 3 compounds (26)(27)(28)(29), the susceptibility to apoptosis appears to be limited to a relatively small fraction of the target cell population in these studies (i.e. between 5 and 15%) (26,27).…”

“…While other laboratories have reported apoptosis in breast tumor cells (including MCF-7 cells) treated with vitamin D 3 compounds (26)(27)(28)(29), the susceptibility to apoptosis appears to be limited to a relatively small fraction of the target cell population in these studies (i.e. between 5 and 15%) (26,27). As a consequence of the prolonged survival of the bulk of the tumor cell population, most studies (both in vitro and in vivo) indicate that l,25-(OH) 2 -D 3 , (28)(29)(30)(31)(32)(33)(34).…”

Mechanisms of the antitumor action of the new antibiotic NC0604, a member of the bleomycin family

“…It is widely used for several malignant tumor types including squamous cell carcinoma, malignant lymphoma, testicular cancer, ovarian cancer, and in combination with other antitumor agents (1). The mechanism of its action is mediated by the generation of reactive oxygen species (ROS), and then degraded DNA and RNA in a selective manner (2,3). The advantage of BLM is that it does not cause suppression of bone marrow and immune system.…”