Purification and properties of DNA topoisomerase II from Daucus carota cells

Topoisomerase II was partially purified from Daucus carota cells by a procedure including ammonium sulphate fractionation, ion-exchange, and affinity chromatography steps. The type II enzyme, identified for its ability to unknot knotted P4 DNA and decatenate Trypanosoma cruzi kDNA, requires ATP and Mg2+ for activity. The unknotting activity was sensitive to an inhibitor of the mammalian type II enzyme, the drug VP16 (IC50 32 mmol m–3), whereas inhibitors of DNA gyrase showed a limited effect on activity. The SDS-PAGE analysis of the dsDNA cellulose fraction revealed the presence of four polypeptides of apparent molecular masses of 72, 71, 34, and 33 kDa among which only a polypeptide of about 70 kDa crossreacted with antibodies against yeast topoisomerase II. Immunoprecipitation experiments with monoclonal antibodies to the and ß isoforms of the human enzyme confirmed the recognition of a polypeptide of 70 kDa. The sedimentation coefficient (S) of the topoisomerase II in the phosphocellulose fraction, calculated by analytical glycerol gradient, was 6.1 corresponding to a molecular mass of about 123 kDa. Results suggest the presence in carrot of a protein of molecular mass of 70 kDa having the typical properties of an eukaryotic topoisomerase II and carrying epitopes recognized by MoAbs to both human and ß enzymes. The 70 kDa polypeptide might then represent the monomer of a homodimer enzyme of 123 kDa.