Knotting of the circular duplex DNA by type II DNA topoisomerase from Drosophila melanogaster.
1983; Elsevier BV; Volume: 258; Issue: 13 Linguagem: Inglês
10.1016/s0021-9258(20)82080-8
ISSN1083-351X
Autores Tópico(s)DNA Repair Mechanisms
ResumoDNA molecules with topological knots can be detected in a reaction mixture of circular DNA and type I1 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 11 requires a high ratio of en- zyme/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 I1 can be efficiently unknotted by a catalytic amount of Drosophila topoisomerase 11, 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.Recent progress in the area of DNA topoisomerases has not only resulted in discovering their ubiquity in a wide range of organisms but has also led to a better understanding of the mechanism by which they convert one DNA topoisomer to another (for review see Refs.1-5).These enzymes are classified into two types according to their mechanisms of action (6, 7 ) .Type I enzymes work by breaking and rejoining single strands in the DNA molecules, while type I1 enzymes work by passing one segment of DNA through another via breaking and rejoining both strands of the DNA molecules.Escherichia coli LJ protein (8), the first topoisomerase discovered, is a prototype of topoisomerase 1 which can remove the negative supercoils efficiently.E. coli DNA gyrase, a very well characterized topoisomerase 11, was found to be able to introduce negative supercoils into covalently closed circular DNA by utilizing the energy from the hydrolysis of ATP (9).Recently several type I1 DNA topoisomerases were discovered and isolated from a variety of sources including bacteriophage T4infected cells (10, l l ) , Xenopus oocytes ( E ) , Drosophila embryos (13), HeLa cell nuclei (14), and yeast (15); these enzymes can catalyze in an ATP-dependent fashion the topoisomerization reactions like relaxation of DNA supercoils, un-
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