Knotting of the circular duplex DNA by type II DNA topoisomerase from Drosophila melanogaster

Abstract

DNA molecules with topological knots can be detected in a reaction mixture of circular DNA and type II DNA topoisomerase purified from the embryos of Drosophila melanogaster. Similar to the DNA knotting reaction by bacteriophage T4-induced topoisomerase (Liu, L. F., Liu, C. C., and Alberts, B. M. (1980) Cell 19, 697-707), the knotting reaction by the Drosophila topoisomerase II requires a high ratio of enzyme/DNA. However, they differ in the following aspects: 1) the knotting reaction by the Drosophila enzyme is greatly stimulated by the presence of ATP; 2) the knotted DNA is a stable product in the reaction mixture over prolonged incubation; and 3) both the covalently closed circular DNA and its nicked form, which is devoid of any superhelical structure, can be knotted. The knotted DNA generated by the Drosophila topoisomerase II can be efficiently unknotted by a catalytic amount of Drosophila topoisomerase II, but not by topoisomerase I. The ionic strength optimum for the knotting reaction is close, but not identical, to the optimal condition for the unknotting and supercoil relaxation reactions. Aspects of DNA knotting are discussed.