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Characterization of the interaction between topoisomerase II and DNA by transcriptional footprinting

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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.

Original languageEnglish
JournalJournal of Molecular Biology
Volume215
Issue2
Pages (from-to)237-44
Number of pages8
ISSN0022-2836
DOIs
Publication statusPublished - 20 Sep 1990

    Research areas

  • Acetanilides, Animals, Base Sequence, Binding Sites, Cattle, DNA, DNA Topoisomerases, Type I, DNA-Binding Proteins, In Vitro Techniques, Molecular Sequence Data, Nitroimidazoles, Transcription, Genetic, Journal Article, Research Support, Non-U.S. Gov't

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