Characterization of the interaction between topoisomerase II and DNA by transcriptional footprinting.
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.
Thomsen
B
B
Department of Molecular Biology and Plant Physiology University of Aarhus, Denmark.
Bendixen
C
C
Lund
K
K
Andersen
A H
AH
Sørensen
B S
BS
Westergaard
O
O
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
D000083:Acetanilides; D004268:DNA-Binding Proteins; D009593:Nitroimidazoles; C054646:Ro 15-0216; D004247:DNA; D004264:DNA Topoisomerases, Type I