原子力显微镜显示，痘苗拓扑异构酶IB以协同方式在DNA上生成细丝。
Atomic force microscopy shows that vaccinia topoisomerase IB generates filaments on DNA in a cooperative fashion.
作者信息
Moreno-Herrero Fernando, Holtzer Laurent, Koster Daniel A, Shuman Stewart, Dekker Cees, Dekker Nynke H
机构信息
Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
出版信息
Nucleic Acids Res. 2005 Oct 19;33(18):5945-53. doi: 10.1093/nar/gki906. Print 2005.
Type IB DNA topoisomerases cleave and rejoin one strand of the DNA duplex, allowing for the removal of supercoils generated during replication and transcription. In addition, electron microscopy of cellular and viral TopIB-DNA complexes has suggested that the enzyme promotes long-range DNA-DNA crossovers and synapses. Here, we have used the atomic force microscope to visualize and quantify the interaction between vaccinia topoisomerase IB (vTopIB) and DNA. vTopIB was found to form filaments on nicked-circular DNA by intramolecular synapsis of two segments of a single DNA molecule. Measuring the filament length as a function of protein concentration showed that synapsis is a highly cooperative process. At high protein:DNA ratios, synapses between distinct DNA molecules were observed, which led to the formation of large vTopIB-induced DNA clusters. These clusters were observed in the presence of Mg2+, Ca2+ or Mn2+, suggesting that the formation of intermolecular vTopIB-mediated DNA synapsis is favored by screening of the DNA charge.
IB型DNA拓扑异构酶切割并重新连接DNA双链中的一条链，从而消除复制和转录过程中产生的超螺旋。此外，对细胞和病毒TopIB-DNA复合物的电子显微镜观察表明，该酶可促进长距离的DNA-DNA交叉和突触形成。在此，我们使用原子力显微镜来可视化和量化痘苗拓扑异构酶IB（vTopIB）与DNA之间的相互作用。发现vTopIB通过单个DNA分子的两个片段的分子内突触作用在带切口的环状DNA上形成细丝。测量细丝长度与蛋白质浓度的关系表明，突触形成是一个高度协同的过程。在高蛋白：DNA比例下，观察到不同DNA分子之间的突触，这导致形成大型vTopIB诱导的DNA簇。在存在Mg2+、Ca2+或Mn2+的情况下观察到这些簇，这表明通过筛选DNA电荷有利于分子间vTopIB介导的DNA突触形成。
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