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

TY - JOUR

T1 - Characterization of the interaction between topoisomerase II and DNA by transcriptional footprinting

AU - Thomsen, B

AU - Bendixen, C

AU - Lund, K

AU - Andersen, A H

AU - Sørensen, B S

AU - Westergaard, O

PY - 1990/9/20

Y1 - 1990/9/20

N2 - 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.

AB - 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.

KW - Acetanilides

KW - Animals

KW - Base Sequence

KW - Binding Sites

KW - Cattle

KW - DNA

KW - DNA Topoisomerases, Type I

KW - DNA-Binding Proteins

KW - In Vitro Techniques

KW - Molecular Sequence Data

KW - Nitroimidazoles

KW - Transcription, Genetic

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/S0022-2836(05)80342-0

DO - 10.1016/S0022-2836(05)80342-0

M3 - Journal article

C2 - 2170662

SN - 0022-2836

VL - 215

SP - 237

EP - 244

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 2

ER -