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

B Thomsen, C Bendixen, K Lund, A H Andersen, B S Sørensen, O Westergaard

    Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

    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.

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

    Keywords

    • 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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