Campbell Spencer, Maxwell Anthony
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK.
J Mol Biol. 2002 Jul 5;320(2):171-88. doi: 10.1016/S0022-2836(02)00461-8.
We have constructed a series of clones encoding N-terminal fragments of human DNA topoisomerase IIalpha. All fragments exhibit DNA-dependent ATPase activity. Fragment 1-420 shows hyperbolic dependence of ATPase on DNA concentration, whereas fragment 1-453 shows hyperstimulation at low ratios of DNA to enzyme, a phenomenon found previously with the full-length enzyme. The minimum length of DNA found to stimulate the ATPase activity was approximately 10 bp; fragments >or=32 bp manifest the hyperstimulation phenomenon. Molecular mass studies show that fragment 1-453 is a monomer in the absence of nucleotides and a dimer in the presence of nucleotide triphosphate. The results are consistent with the role of the N-terminal domain of topoisomerase II as an ATP-operated clamp that dimerises in the presence of ATP. The hyperstimulation effect can be interpreted in terms of a "piggy-back binding" model for protein-DNA interaction.
我们构建了一系列编码人DNA拓扑异构酶IIα N端片段的克隆。所有片段均表现出依赖DNA的ATP酶活性。片段1 - 420的ATP酶活性对DNA浓度呈双曲线依赖性，而片段1 - 453在低DNA与酶比例时表现出过度刺激，这一现象先前在全长酶中也有发现。发现能刺激ATP酶活性的DNA最短长度约为10 bp；长度≥32 bp的片段表现出过度刺激现象。分子量研究表明，片段1 - 453在无核苷酸时为单体，在有三磷酸核苷酸时为二聚体。这些结果与拓扑异构酶II的N端结构域作为一个在ATP存在下二聚化的ATP操作钳的作用相一致。过度刺激效应可以用蛋白质 - DNA相互作用的“搭便车结合”模型来解释。
