Characterization of a topoisomerase-like activity at specific hypersensitive sites in the Drosophila histone gene cluster

It is well known that treatment of DNA-topoisomerase complexes with SDS induces cleavage of the DNA by trapping a reactive intermediate in which the topoisomerase is covalently linked to the terminal phosphates of the cut DNA. I have used this technique to examine potential topoisomerase binding sites in the histone gene chromatin of Drosophila Kc cells. Treatment of Kc nuclei with SDS induces Mg++-dependent DNA cleavage near the borders of two nuclease-hypersensitive sites located 5' and 3' of histone H4. It is likely that the SDS-induced cleavage at these hypersensitive sites is due to a topoisomerase because protein becomes tightly bound to the ends of the cleaved DNA fragments. Preliminary experiments suggest that a type II topoisomerase may be responsible for the cleavage.     

The dynamics of chromatin condensation: redistribution of topoisomerase II in the 87A7 heat shock locus during induction and recovery.

We have examined the in vivo sites of action for topoisomerases II in the 87A7 heat shock locus as a function of gene activity. When the hsp70 genes are induced, there is a dramatic redistribution of topoisomerase II in the locus which parallels many of the observed alterations in chromatin structure. In addition to changes in the topoisomerase II distribution within the locus, we find topoisomerase II localized around the putative domain boundaries scs and scs'. During recovery, when the chromatin fiber of the locus recondenses, the major sites of action for topoisomerase II appear to be located within the two hsp70 genes and in the intergenic spacer separating the two genes.     

Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding

The sequence dependence of Drosophila topoisomerase II supercoil relaxation and binding activities has been examined. The DNA substrates used in binding experiments were two fragments from Drosophila heat shock locus 87A7. One of these DNA fragments includes the coding region for the heat shock protein hsp70, and the other includes the intergenic non-coding region that separates two divergently transcribed copies of the hsp70 gene at the locus. The intergenic region was previously shown to have a much higher density of topoisomerase cleavage sites than the hsp70 coding region. Competition nitrocellulose filter binding assays demonstrate a preferential binding of the intergene fragment, and that binding specificity increases with increasing ionic strength. Dissociation kinetics indicate a greater kinetic stability of topoisomerase II complexes with the intergene DNA fragment. To study topoisomerase II relaxation activity, we used supercoiled plasmids that contained the same fragments from locus 87A7 cloned as inserts. The relative relaxation rates of the two plasmids were determined under several conditions of ionic strength, and when the plasmid substrates were included in separate reactions or when they were mixed in a single reaction. The relaxation properties of these two plasmids can be explained by a coincidence of high-affinity binding sites, strong cleavage sites, and sites used during the catalysis of strand passage events by topoisomerase II. Sequence dependence of topoisomerase II catalytic activity may therefore parallel the sequence dependence of DNA cleavage by this enzyme.