DNA topoisomerase I1 was purified from calf thymus nuclei by a simple and fast four-step procedure:selective ammonium sulfate precipitation, chromatography on blue-Sepharose and hydroxyapatite, followed by ultracentrifugation on a glycerol gradient. Starting from 300 g thymus glands, this procedure yields 0.7 mg of homogeneous topoisomerase 11. The final product is free of any nucleolytic, proteolytic or topoisomerase I activity. Dodecylsulfate/polyacrylamide gel electrophoresis reveals two bands with apparent molecular masses of 175 and 150 kDa. Analytical gel filtration and sedimentation on isokinetic sucrose gradients were used to determine the Stokes' radius as 6.4 nm and the sedimentation coefficient as 9.5 S, indicating a dimeric structure for the native enzyme. The purified topoisomerase I1 is strictly dependent on ATP or dATP, the K,,, values of which were 0.14 mM and 0.5 mM, respectively. Mg2+ is an essential cofactor for the reaction at concentrations between 0.5 -8 mM, with an optimum at 4 mM. Mg2+ can be substituted by Mn2+ at concentrations between 0.2-0.4 mM. Both the relaxation and the catenation reaction exhibit a salt optimum at 130 mM NaC1. At concentrations below 30 mM and above 200 mM, the enzyme is inactive. The pH is optimal between 8 and 9.5 using Tris buffers.DNA topoisomerases control and modify the topological state of DNA. Two classes of topoisomerases, type I and type 11, are known to exist in prokaryotes [l, 21 and eukaryotes [3, 41. The type I enzymes are thought to relieve torsional constraints in DNA by making a transient single-strand nick, allowing the rotation of one DNA strand around the other.By contrast, type I1 topoisomerases transiently break a pair of complementary DNA strands and pass another doublestranded segment. Thus, type I1 enzymes can catalyze many types of interconversions between different topological forms of DNA. Examples are catenation and decatenation, knotting and unknotting.The importance of topoisomerase I1 in bacteria and phages has become clear during the recent years (reviewed in [5 -101). The function of type I1 enzymes in eukaryotes, however, is less well understood. The localization of topoisomerase I1 at the nuclear matrix [l 11 points to a functional role in gene expression. The observation that DNA breakage produced by a manifold of antitumor drugs is mediated by mammalian topoisomerase I1 suggests a function in sister chromatid exchange and thus in DNA repair [12-141. The observation that the level of topoisomerase 11, but not that of topoisomerase I, is stimulated more than tenfold by cell proliferation [15, 161 points to a primary role of this enzyme in the process of DNA replication. Furthermore, it has been Correspondence to F. Grosse, Abteilung Chemie,
