title: Bimodal recognition of DNA geometry by human topoisomerase II alpha: preferential relaxation of positively supercoiled DNA requires elements in the C-terminal domain.
abstract: Human topoisomerase IIalpha, but not topoisomerase IIbeta, can sense the geometry of DNA during relaxation and removes positive supercoils >10-fold faster than it does negative superhelical twists. In contrast, both isoforms maintain lower levels of DNA cleavage intermediates with positively supercoiled substrates. Since topoisomerase IIalpha and IIbeta differ primarily in their C-terminal domains (CTD), this portion of the protein may play a role in sensing DNA geometry. Therefore, to more fully assess the importance of the topoisomerase IIalpha CTD in the recognition of DNA topology, hTop2alphaDelta1175, a mutant human enzyme that lacks its CTD, was examined. The mutant enzyme relaxed negative and positive supercoils at similar rates but still maintained lower levels of cleavage complexes with positively supercoiled DNA. Furthermore, when the CTD of topoisomerase IIbeta was replaced with that of the alpha isoform, the resulting enzyme preferentially relaxed positively supercoiled substrates. In contrast, a chimeric topoisomerase IIalpha that carried the CTD of the beta isoform lost its ability to recognize the geometry of DNA supercoils during relaxation. These findings demonstrate that human topoisomerase IIalpha recognizes DNA geometry in a bimodal fashion, with the ability to preferentially relax positive DNA supercoils residing in the CTD. Finally, results with a series of human topoisomerase IIalpha mutants suggest that clusters of positively charged amino acid residues in the CTD are required for the enzyme to distinguish supercoil geometry during DNA relaxation and that deletion of even the most C-terminal cluster abrogates this recognition.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
authors.forename: A Kathleen
authors.initials: AK
authors.lastname: McClendon
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Gentry
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Dickey
authors.forename: Marie
authors.initials: M
authors.lastname: Brinch
authors.forename: Simon
authors.initials: S
authors.lastname: Bendsen
authors.forename: Anni H
authors.initials: AH
authors.lastname: Andersen
authors.forename: Neil
authors.initials: N
authors.lastname: Osheroff
mesh_terms: D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D053837:DNA Cleavage; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000235:genetics; D017384:Sequence Deletion; D059005:Topoisomerase II Inhibitors
chemical_list: D000951:Antigens, Neoplasm; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D059005:Topoisomerase II Inhibitors; D004250:DNA Topoisomerases, Type II