title: Regulation of the function of eukaryotic DNA topoisomerase I: analysis of the binding step and of the catalytic constants of topoisomerization as a function of DNA topology.
abstract: It was previously observed that two steps of the reaction of eukaryotic DNA topoisomerase I (topoisomerization and cleavage) depend upon the conformation of the DNA substrate: in both instances the supercoiled form is a more efficient substrate than the relaxed one. This paper reports the analysis of two other steps of the reaction: the binding of DNA topoisomerase I to DNA and the catalytic constants (Kcs) of topoisomerization as a function of the topology of the substrate. Binding. Competition assays show that supercoiled DNA binds the enzyme with even slower kinetics than the relaxed form. Therefore, the preferential topoisomerization of supercoiled DNA is not due to the binding step. Additional evidence that the rate-limiting step of the topoisomerization reaction is not the binding of the enzyme to DNA is provided by the fact that the kinetics of relaxation is first order. Catalysis. The Kcs of the topoisomerization reaction have been calculated and it was shown that they do not vary as a function of the topology of the substrate or of its size. Taken together, the data on binding, cleavage, topoisomerization, and Kcs suggest that the preferential topoisomerization of torsionally strained DNA is due to the higher availability, on this topological form, of DNA sites that allow the onset of the reaction.
authors.affiliation: Centro Studi Acidi Nucleici (CNR), Rome, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Caserta
authors.forename: A
authors.initials: A
authors.lastname: Amadei
authors.forename: G
authors.initials: G
authors.lastname: Camilloni
authors.forename: E
authors.initials: E
authors.lastname: Di Mauro
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002384:Catalysis; D004264:DNA Topoisomerases, Type I / Q000502:physiology*; D004270:DNA, Circular / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding
chemical_list: D004270:DNA, Circular; D004278:DNA, Superhelical; D004264:DNA Topoisomerases, Type I