Thomsen B, Bendixen C, Lund K, Andersen A H, Sørensen B S, Westergaard O
Department of Molecular Biology and Plant Physiology University of Aarhus, Denmark.
J Mol Biol. 1990 Sep 20;215(2):237-44. doi: 10.1016/S0022-2836(05)80342-0.
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.
利用转录分析法对小牛胸腺拓扑异构酶II与DNA之间的相互作用进行了表征。在噬菌体RNA聚合酶特异性启动子下游的任一方向插入了一个拓扑异构酶II的高度优先识别序列。模板上拓扑异构酶II-DNA复合物的存在引发转录受阻，产生在拓扑异构酶II结合位点5'端终止的RNA转录本。从两侧向复合物进行转录得到的拓扑异构酶II足迹表明，真核拓扑异构酶II结合一个28个碱基对的区域，其中心有一个22个碱基对的高度受保护核心。结合区域围绕拓扑异构酶II介导的切割位点对称定位。与该结果一致，使用由与受保护区域同源的28聚体寡核苷酸组成的DNA底物观察到了最佳的拓扑异构酶II介导的切割。从28聚体末端逐步去除碱基对逐渐降低了酶介导的切割水平。
