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Drug-specific Sites of Topoisomerase II DNA Cleavage in Drosophila Chromatin: Heterogeneous Localization and Reversibility¹

Division of Experimental Oncology B, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan, Italy

DNA cleavage stimulated by different topoisomerase II inhibitors shows in vitro a characteristic sequence specificity. Since chromatin structure and genome organization are expected to influence drug-enzyme interactions and repair of drug-induced DNA lesions, we investigated topoisomerase II DNA cleavage sites stimulated by teniposide (VM-26), 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin (dh-EPI, a doxorubicin derivative), 4'-(9-acridinylamino)-methanesulfon-m-anisidide, and amonafide in the histone gene locus and satellite III DNA of Drosophila cells with Southern blottings and genomic sequencing by primer extension. VM-26 stimulated cleavage in the satellite III DNA, whereas the other studied drugs did not. All four drugs stimulated cleavage in the histone gene cluster, but they yielded drug-specific cleavage intensity patterns. Cleavage sites by dh-EPI and VM-26 were sequenced in the histone H2A gene promoter and were shown to be distinct. DNA cleavage analysis in cloned DNA fragments with Drosophila topoisomerase II showed that drugs stimulated the same sites in vivo and in vitro. Strand cuts were in vivo staggered by 4 bases, and base sequences at major dh-EPI and VM-26 sites completely agreed with known in vitro drug sequence specificities. Moreover, DNA cleavage reverted faster in the satellite III than in the histone repeats. While stimulating similar levels of DNA breakage in bulk genomic DNA, dh-EPI and VM-26 markedly differed for cleavage extent and reversibility in specific chromatin loci. The results demonstrate a high heterogeneity in the localization, extent, and reversibility of drug-stimulated DNA cleavage in the chromatin of living cells.

¹ This work was supported partially by the Associazione Italiana per la Ricerca sul Cancro (Milan); by the Consiglio Nazionale delle Ricerche, Progetto Finalizzato Applicazioni Cliniche della Ricerca Oncologica (Rome); and by the Ministero della Sanità (Rome). E. K. received grant support from the Association pour la Recherche sur le Cancer.

² Present address: Laboratoire de Biologie Moléculaire Eucaryote, CNRS, Toulouse, 31062 France.

³ To whom requests for reprints should be addressed, at Division of Experimental Oncology B, Istituto Nazionale Tumori, 20133 Milan, Italy. Phone: 39-2-2390-203; Fax: 39-2-2390-764; E-mail: capranico@icil64.cilea.it.

Received 11/ 3/95. Accepted 2/14/96.

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