title: The role of DNA bending in type IIA topoisomerase function.
abstract: Type IIA topoisomerases control DNA supercoiling and separate newly replicated chromosomes using a complex DNA strand cleavage and passage mechanism. Structural and biochemical studies have shown that these enzymes sharply bend DNA by as much as 150°; an invariant isoleucine, which has been seen structurally to intercalate between two base pairs outside of the DNA cleavage site, has been suggested to promote deformation. To test this assumption, we examined the role of isoleucine on DNA binding, bending and catalytic activity for a bacterial type IIA topoisomerase, Escherichia coli topoisomerase IV (topo IV), using a combination of site-directed mutagenesis and biochemical assays. Our data show that alteration of the isoleucine (Ile172) did not affect the basal ATPase activity of topo IV or its affinity for DNA. However, the amino acid was important for DNA bending, DNA cleavage and supercoil relaxation. Moreover, an ability to bend DNA correlated with efficacy with which nucleic acid substrates stimulate ATP hydrolysis. These data show that DNA binding and bending by topo IV can be uncoupled, and indicate that the stabilization of a highly curved DNA geometry is critical to the type IIA topoisomerase catalytic cycle.
authors.affiliation: Department of Molecular and Cell Biology, MC 3220 University of California, Berkeley, CA 94720-3220, USA.
authors.forename: Imsang
authors.initials: I
authors.lastname: Lee
authors.forename: Ken C
authors.initials: KC
authors.lastname: Dong
authors.forename: James M
authors.initials: JM
authors.lastname: Berger
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D019943:Amino Acid Substitution; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D053837:DNA Cleavage; D027101:DNA Topoisomerase IV / Q000737:chemistry* / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D007532:Isoleucine / Q000737:chemistry; D009690:Nucleic Acid Conformation
chemical_list: D004278:DNA, Superhelical; D007532:Isoleucine; D004247:DNA; D000251:Adenosine Triphosphatases; D027101:DNA Topoisomerase IV