Isolation of intercalator-dependent protein-linked DNA strand cleavage activity from cell nuclei and identification as topoisomerase II.
DNA intercalating agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) have previously been found to induce in mammalian cells the formation of protein-associated DNA single- and double-strand breaks. In the current work, an activity characterized by the production of DNA-protein links associated with DNA strand breaks and by stimulation by m-AMSA was isolated from L1210 cell nuclei and was shown to be due to topoisomerase II. Nuclei were extracted with 0.35 M NaCl, and the extract was fractionated by gel filtration, DNA-cellulose chromatography, and glycerol gradient centrifugation. A rapid filter binding assay was devised to monitor the fractionation procedure on the basis of DNA-protein linking activity. The active DNA-cellulose fraction contained both topoisomerase I and topoisomerase II whereas the glycerol gradient purified material contained only topoisomerase II activity. The properties of the active material were studied at both stages of purification. m-AMSA enhanced the formation of complexes between purified topoisomerase II and SV40 DNA in which the DNA sustained a single- or double-strand cut and the enzyme was covalently linked to the 5' terminus of the DNA. This action was further enhanced by ATP, as well as by nonhydrolyzable ATP analogues. m-AMSA inhibited the topoisomerization and catenation reactions of topoisomerase II, probably because of trapping of the enzyme-DNA complexes. The activity showed a dependence on the type of DNA intercalators used, analogous to what was previously observed in intact cells. m-AMSA had no effect on topoisomerase I.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D002846:Chromatography, Affinity; D004250:DNA Topoisomerases, Type II / Q000302:isolation & purification* / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D007364:Intercalating Agents / Q000494:pharmacology*; D007700:Kinetics; D007939:Leukemia L1210 / Q000201:enzymology*; D051379:Mice; D011485:Protein Binding; D013539:Simian virus 40; D013816:Thermodynamics
D004279:DNA, Viral; D007364:Intercalating Agents; D004250:DNA Topoisomerases, Type II