ANALYSIS OF TOPOISOMERASE II-MEDIATED DNA CLEAVAGE IN THE 5'-REGION OF THE DROSOPHILA HSP70 GENE - IDENTIFICATION OF A NOVEL HALF-SITE DNA SUBSTRATE FOR TOPOISOMERASE-II CLEAVAGE

Previous in vivo studies have identified a prominent oxin-9-(4,6-O-thi onylidine-beta-D-glucopyranoside) (VM-26)-induced double-stranded topo isomerase II cleavage site at almost-equal-to +80 relative to the star t of Drosophila hsp70 transcription (Kroeger, P. E., and Rowe, T. C. ( 1992) Biochemistry 31, 2492-2502). Topoisomerase II binding at this si te correlated with the repression of hsp70 transcription suggesting th at this protein-DNA interaction was important in the regulation of hsp 70 gene expression. In this paper, we investigated the interaction of purified Drosophila topoisomerase II with a 271 -base pair DNA fragmen t containing the +80 region of the hsp70 gene using the topoisomerase II-specific inhibitor VM-26. VM-26-induced topoisomerase II cleavage o f the hsp70 DNA resulted in a major 4-base staggered double-stranded b reak at +84. In the absence of ATP the +84 site was the only significa nt VM-26-induced cleavage site. Addition of ATP to the reaction result ed in a stimulation of topoisomerase cleavage throughout the 271-base pair DNA fragment. Deletion analyses determined that almost-equal-to 1 5 to 25 bp of flanking sequence were required for efficient cleavage a t most topoisomerase II sites within the hsp70 DNA. However, in the ca se of the +84 site, topoisomerase cleavage still occurred even when th is site was split in half by the restriction enzyme PstI. Topoisomeras e II cleavage of both ''half-site'' DNA molecules occurred at the corr ect positions on the 4-base single-stranded DNA overhangs generated by PstI. Cleavage was reversible indicating that topoisomerase II could reseal the single-stranded DNA break formed in each half-site substrat e. Denaturation of the half-site molecules abolished topoisomerase II cleavage suggesting that cleavage required the duplex region adjacent to the single-stranded cleavage site. Identification of this unusual h alf-site substrate provides additional evidence that double-stranded c leavage of DNA by topoisomerase II occurs via two sequential single-st randed breaks.