DNA topoisomerase II from Drosophila melanogaster. Purification and physical characterization.

Abstract

A type I1 DNA topoisomerase has been purified from the nuclei of Drosophila melanogaster 6-to 18-h-old embryos.The enzyme, as assayed by its ability to catenate supercoiled DNA, behaved as a single homogeneous species throughout the procedure and the yield was approximateiy 0.5 mg of protein/100 g of dechorionated embryos.The final product was entirely ATPdependent and free of topoisomerase I, endonucIease and protease activities.Th? purified topoisomerase I1 had a Stokes radius of 69 A and a sedimentation coefficient (sz0,J of 9.2 S, leading to a calculated native molecular weight of approximately 261,000.The protein consists of a single polypeptide of molecular weight 166,000, as determined by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels.Taken together with the above hydrodynamic studies, the Drosophila enzyme is probably a homodimer, as has been observed for other eukaryotic type I1 enzymes.Thus, it appears that during the course of evolution the heterologous subunits which comprise bacterial type I1 topoisomerases have been combined into a single polypeptide chain in eukaryotes.Topoisomerases are a class of enzymes able to interconvert various topological forms of DNA without altering the primary structure of the DNA.Among the reactions catalyzed by these enzymes are relaxation-supercoiling, knotting-unknotting and catenation-decatenation of DNA (for reviews, see Refs.1-5).Mechanistically, topoisomerases fall into two classes.The t-ype I enzymes, such as Escherichia coli w protein, are able to relieve torsional constraints in DNA by making a transient single-stranded nick, allowing the rotation of one DNA strand around the other.In contrast, type I1 topoisomerases alter the topology of DNA by passing one DNA helix through a transient double-stranded break in another helical segment.This was first demonstrated for E. coli DNA gyrase, which can both induce and remove negative supercoils in closed circular duplex DNA using this double-strand passage mechanism (6).Type I1 and type I topoisomerases are also distinguished by the absolute requirement of the type I1 enzymes for ATP.