title: Characterization of the interaction between topoisomerase II and DNA by transcriptional footprinting.
abstract: The interaction between calf thymus topoisomerase II and DNA has been characterized using a transcription assay. A highly preferred recognition sequence for topoisomerase II was inserted in either direction downstream from a promoter specific for a bacteriophage RNA polymerase. The presence of topoisomerase II-DNA complexes on the template provoked blockage of transcription, yielding RNA transcripts terminated 5' to the topoisomerase II binding site. A footprint of topoisomerase II, derived from transcription towards the complex from either side, revealed that eukaryotic topoisomerase II binds a region of 28 base-pairs with a highly protected central core of 22 base-pairs. The binding region was located symmetrically around the topoisomerase II-mediated cleavage site. In agreement with this result, optimal topoisomerase II-mediated cleavage was observed with a DNA substrate consisting of a 28-mer oligonucleotide homologous to the protected region. Stepwise removal of base-pairs from the ends of the 28-mer gradually reduced the level of enzyme-mediated cleavage.
authors.affiliation: Department of Molecular Biology and Plant Physiology University of Aarhus, Denmark.
authors.forename: B
authors.initials: B
authors.lastname: Thomsen
authors.forename: C
authors.initials: C
authors.lastname: Bendixen
authors.forename: K
authors.initials: K
authors.lastname: Lund
authors.forename: A H
authors.initials: AH
authors.lastname: Andersen
authors.forename: B S
authors.initials: BS
authors.lastname: Sørensen
authors.forename: O
authors.initials: O
authors.lastname: Westergaard
mesh_terms: D000083:Acetanilides / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009593:Nitroimidazoles / Q000494:pharmacology; D014158:Transcription, Genetic
chemical_list: D000083:Acetanilides; D004268:DNA-Binding Proteins; D009593:Nitroimidazoles; C054646:Ro 15-0216; D004247:DNA; D004264:DNA Topoisomerases, Type I