Rhodobacter capsulatus DNA topoisomerase I purification and characterization

A 30-kDa DNA topoisomerase has been purified to near homogeneity from the p urple nonsulfur photosynthetic bacterium Rhodobacter capsulatus. The enzyme is recognized by an antibody against a 16-mer peptide sequence from human DNA topoisomerase I. The purified enzyme is a type I topoisomerase. Consist ent with the properties of other prokaryotic type I DNA topoisomerases, the isolated enzyme is unable to relax positively supercoiled DNA and absolute ly requires divalent cations for its relaxation activity. However, regardle ss of the Mg+2 concentrations, ATP concentrations above 5 mM completely inh ibit the relaxing activity. The enzyme is sensitive to high salt concentrat ions and the optimal activity occurs at salt concentrations between 3 and 3 0 mM for monovalent cations. Single-stranded M13 DNA is a strong inhibitor of this relaxing activity. The enzyme is inhibited by ethidium bromide, con firming that this DNA topoisomerase is incapable of relaxing positive super coils. Topoisomerase I-specific inhibitors like Hoechst 32258 and actinomyc in D inhibit the enzymatic activity while the enzyme is resistant to type I I topoisomerase inhibitors such as norfloxacin, nalidixic acid, and novobio cin. From these enzymatic characteristics, we conclude that the R. capsulat us DNA topoisomerase is a prokaryotic type I DNA topoisomerase. (C) 1999 Ac ademic Press.