ABSTRACT
DNA topoisomerase I has been purified from Mycobacterium smegmatis to near homogeneity using different column chromatographic techniques. The enzyme activity relaxes form I DNA into form IV DNA, requiring Mg2+, but not ATP or any other cofactors for its activity. Several properties of the enzyme were found to be similar to that of the prototype enzyme, Escherichia coli topoisomerase I.
Subject(s)
DNA Topoisomerases, Type I/isolation & purification , Mycobacterium/enzymologyABSTRACT
A type 1 DNA topoisomerase has been purified from the nuclei of the kinetoplast hemoflagellate Leishmania donovani using polyethylene glycol fractionation and chromatography on hydroxylapatite, phosphocellulose and phenylsepharose column. The relaxation activity is ATP independent. Mg2+ is an essential cofactor for the reaction with an optimum at 10 mM. Mg2+ can be substituted by Mn2+ at 5 mM concentration. The relaxation reaction exhibits a salt optimum at 100 mM KCl. The enzyme can not remove supercoils from positive superhelical DNAs nor can induce supercoiling of relaxed DNAs. The topoisomerase activity is associated with a polypeptide of molecular weight about 67 kDa as shown by sephacryl-S200 gel filtration and by electrophoresis on sodium dodecyl sulphate-polyacrylamide gels.