DRUG-SPECIFIC SITES OF TOPOISOMERASE-II DNA CLEAVAGE IN DROSOPHILA CHROMATIN - HETEROGENEOUS LOCALIZATION AND REVERSIBILITY

DNA cleavage stimulated by different topoisomerase II inhibitors shows in vitro a characteristic sequence specificity, Since chromatin struc ture and genome organization are expected to influence drug-enzyme int eractions and repair of drug-induced DNA lesions, we investigated topo isomerase II DNA cleavage sites stimulated by teniposide (VM-26), deme thoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin (dh-EPI, a doxorubicin derivative), 4'-(9-acridinylamino)-methanesulfon-m-anisidide and amona fide in the histone gene locus and satellite III DNA of Drosophila cel ls 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 hist one gene cluster, but they yielded drug-specific cleavage intensity pa tterns, Cleavage sites by dh-EPI and VM-26 were sequenced in the histo ne H2A gene promoter and were shown to be distinct, DNA cleavage analy sis in cloned DNA fragments with Drosophila topoisomerase II showed th at drugs stimulated the same sites ill vivo and in vitro. Strand cuts were ira vivo staggered by 4 bases, and base sequences at major dh-EPI and VM-26 sites completely agreed with known in vitro drug sequence s pecificities. Moreover, DNA cleavage reverted faster in the satellite III than in the histone repeats, While stimulating similar levels of D NA breakage in bulk genomic DNA, dh-EPI and VM-26 markedly differed fo r cleavage extent and reversibility in specific chromatin loci, The re sults demonstrate a high heterogeneity in the localization, extent, an d reversibility of drug-stimulated DNA cleavage in the chromatin of li ving cells.