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Irreversible and Reversible Topoisomerase II DNA Cleavage Stimulated by Clerocidin: Sequence Specificity and Structural Drug Determinants¹

Division of Experimental Oncology B, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan, Italy [M. B., F. Z., R. F., G. C.], and Department of Pharmaceutical Sciences, University of Padua, 35131 Padua, Italy [G. Z. M. P.]

In contrast to other topoisomerase II poisons, the microbial terpenoid clerocidin was shown to stimulate irreversible topoisomerase II-mediated DNA cleavage. To establish the structural determinants for drug activity, in this study we have investigated intensity patterns and sequence specificity of clerocidin-stimulated DNA cleavage using 5'-end ³²P-labeled DNA fragments. At a majority of the sites, clerocidin-stimulated cleavage did not revert upon NaCl addition; nevertheless, at some sites, cleavage completely reverted. Statistical analyses showed that drug-preferred bases were different in the two cases: guanine and cytosine were highly preferred at position -1 at irreversible and reversible sites, respectively. These results demonstrated that cleavage irreversibility was site selective and required a guanine at the 3' end of the cut. Further experiments revealed that some irreversible sites showed an abnormal electrophoretic mobility in sequencing gels with respect to cleaved bands generated by 4-(9-acridinylamino)methanesulfon-m-anisidide, suggesting a chemical alteration of the DNA strand. Interestingly, 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 clerocidin may react with the DNA (guanine at -1) in the ternary complex, resulting in cleavage irreversibility and in altered DNA mobility in sequencing gels.

¹ This work has been supported in part by the Associazione Italiana per la Ricerca sul Cancro, Milan, Italy, and the Consiglio Nazionale delle Ricerche, Progetto Finalizzato Applicazioni Cliniche della Ricerca Oncologica, Rome, Italy.

² To whom requests for reprints should be addressed. Phone: 39-2-2390203; Fax: 39-2-2390764; E-mail: capranico@istitutotumori.mi.it.

Received 10/23/96. Accepted 3/ 1/97.

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