amonafide in the histone gene locus and satellite III DNA of Drosophila

DNA cleavage stimulated by different topolsomerase H inhibitors shows in vitro a characteristic sequence specificity. Since chromatin struc tare 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 doxo rubicin 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 genomicsequencingby primer exten sion. VM-26 stimulated cleavage in the satellite Ill 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 deavage reverted faster in the satellite ifi 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 reversibifity 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.