Abstract
The purified type II DNA topoisomerase from the embryos of Drosophila melanogaster exists in its native form as a dimer of 170,000-dalton polypeptides. In addition to the 170,000-dalton polypeptides, 3 polypeptides with molecular weights of 151,000, 141,000, and 132,000 were resolved when the enzyme was analyzed by electrophoresis under denaturing conditions. All four polypeptides can participate in the topoisomerase cleavage reaction and form covalent complexes with the cleaved DNA. Furthermore, immunochemical and biochemical data showed that they are structurally related and, therefore, the smaller polypeptides are likely generated from the 170,000-dalton polypeptide by proteolysis. The double strand DNA cleavage reaction of Drosophila topoisomerase has different site specificity from the Escherichia coli DNA gyrase-effected reaction. However, they result in an identical DNA structure at the cleavage site, which is a staggered double strand break with 4-nucleotide long 5'-protruding ends. The 3'-ends at the site of cleavage by Drosophila topoisomerase II have free hydroxyl groups and can be extended by exactly 4 nucleotides with T4 DNA polymerase, while the 5'-ends are covalently linked to the topoisomerase molecules. This similarity in cleavage site structure for Drosophila topoisomerase II and E. coli DNA gyrase suggests that they share some fundamental features in their mechanism of action.
