IRREVERSIBLE AND REVERSIBLE TOPOISOMERASE-II DNA CLEAVAGE STIMULATED BY CLEROCIDIN - SEQUENCE SPECIFICITY AND STRUCTURAL DRUG DETERMINANTS

In contrast to other topoisomerase II poisons, the microbial terpenoid clerocidin was shown to stimulate irreversible topoisomerase II-media ted DNA cleavage. To establish the structural determinants for drug ac tivity, in this study we have investigated intensity patterns and sequ ence specificity of clerocidin-stimulated DNA cleavage using 5'-end P- 32-labeled DNA fragments. At a majority of the sites, clerocidin-stimu lated cleavage did not revert upon NaCl addition; nevertheless, at som e sites, cleavage completely reverted. Statistical analyses showed tha t drug-preferred bases were different in the two cases: guanine and cy tosine were highly preferred at position -1 at irreversible and revers ible sites, respectively. These results demonstrated that cleavage irr eversibility was site selective and required a guanine at the 3' end o f the cut. Further experiments revealed that some irreversible sites s howed an abnormal electrophoretic mobility in sequencing gels with res pect to cleaved bands generated by 4-(9-acridinylamino)methanesulfon-m -anisidide, suggesting a chemical alteration of the DNA strand. Intere stingly, the ability to stimulate irreversible cleavage progressively decreased over time when clerocidin was stored in ethanol. Under these conditions, nuclear magnetic resonance measurements demonstrated that the drug underwent structural modifications that involved the C-12-C- 15 side chain. Thus, the results indicate that a specific moiety of cl erocidin may react with the DNA (guanine at -1) in the ternary complex , resulting in cleavage irreversibility and in altered DNA mobility in sequencing gels.