Interactions of Drosophila DNA topoisomerase II with left-handed Z-DNA in supercoiled minicircles.
The native form of Drosophila melanogaster DNA topoisomerase II was purified from Schneider's S3 tissue culture cells and studied with two supercoiled minicircle preparations, mini and mini-CG, 354 bp and 370 bp in length, respectively. Mini-CG contains a d(CG)7 insert which assumes a left-handed Z-DNA conformation in negative supercoiled topoisomers with a negative linking number difference - delta Lk greater than or equal to 2. The interactions of topoisomerase II with topoisomer families of mini and mini-CG were studied by band-shift gel electrophoresis in which the individual topoisomers and their discrete or aggregated protein complexes were resolved. A monoclonal anti-Z-DNA IgG antibody (23B6) bound and aggregated only mini-CG, thereby confirming the presence of Z-DNA. Topoisomerase II bound and relaxed mini-CG more readily than mini. In both cases, there was a preference for more highly negatively supercoiled topoisomers. The topoisomerase II inhibitor VM-26 induced the formation of stable covalent DNA-protein intermediates. In addition, the non-hydrolyzable GTP analogue GTP gamma S inhibited the binding and relaxation activities. Experiments to detect topoisomerase cleavage sites failed to elicit specific loci on either minicircle preparation. We conclude that Drosophila topoisomerase II is able to bind and process small minicircles with lengths as short as 360 bp and negative superhelix densities, - sigma, which can exceed 0.1. Furthermore, the enzyme has a preferential affinity for topoisomers containing Z-DNA segments and relaxes these molecules, presumably by cleavage external to the inserts. Thus, a potentially functional relationship between topoisomerase II, an enzyme regulating the topological state of DNA-chromatin in vivo, and left-handed Z-DNA, a conformation stabilized by negative supercoiling, has been established.
Glikin
G C
GC
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, FRG.
Jovin
T M
TM
Arndt-Jovin
D J
DJ
D000818:Animals; D000911:Antibodies, Monoclonal; D004250:DNA Topoisomerases, Type II / Q000187:drug effects / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D004586:Electrophoresis; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D013713:Teniposide / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D004270:DNA, Circular; D004278:DNA, Superhelical; D011089:Polydeoxyribonucleotides; C019530:poly(dC-dG); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013713:Teniposide; D004250:DNA Topoisomerases, Type II