Isolation of intercalator-dependent protein-linked DNA strand cleavage activity from cell nuclei and identification as topoisomerase II

Biochemistry. 1986 Jan 14;25(1):9-16. doi: 10.1021/bi00349a002.

Abstract

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)

MeSH terms

  • Animals
  • Cell Nucleus / enzymology*
  • Chromatography, Affinity
  • DNA Topoisomerases, Type II / isolation & purification*
  • DNA Topoisomerases, Type II / metabolism
  • DNA, Viral / metabolism
  • Intercalating Agents / pharmacology*
  • Kinetics
  • Leukemia L1210 / enzymology*
  • Mice
  • Protein Binding
  • Simian virus 40
  • Thermodynamics

Substances

  • DNA, Viral
  • Intercalating Agents
  • DNA Topoisomerases, Type II