Peng H, Marians K J
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
J Biol Chem. 1995 Oct 20;270(42):25286-90. doi: 10.1074/jbc.270.42.25286.
The two type II topoisomerases in Escherichia coli, DNA gyrase and topoisomerase (Topo) IV, share considerable amino acid sequence similarity, yet they have distinctive topoisomerization activities. Only DNA gyrase can supercoil relaxed DNA, whereas during oriC DNA replication in vitro, only Topo IV can support the final stages of replication, processing of the late intermediate and decatenation of the daughter molecules. In order to develop an understanding for the basis of the differential activities of these two enzymes, we have initiated a characterization of Topo IV binding to DNA. We find that unlike gyrase, Topo IV neither constrains DNA in a positive supercoil when it binds nor protects a 150-base pair region of DNA from digestion with micro-coccal nuclease. Consistent with this, DNase I footprinting experiments showed that Topo IV protected a 34-base pair region roughly centered about the topoisomerase-induced cleavage site. In addition, Topo IV preferentially bound supercoiled rather than relaxed DNA. Thus, the DNA binding characteristics of Topo IV are more akin to those of the type II eukaryotic enzymes rather than those of its prokaryotic partner.
大肠杆菌中的两种II型拓扑异构酶，即DNA促旋酶和拓扑异构酶IV（Topo IV），具有相当高的氨基酸序列相似性，但它们具有独特的拓扑异构化活性。只有DNA促旋酶能够使松弛的DNA超螺旋化，而在体外oriC DNA复制过程中，只有Topo IV能够支持复制的最后阶段、后期中间体的加工以及子代分子的解连环。为了深入了解这两种酶差异活性的基础，我们开始对Topo IV与DNA的结合进行表征。我们发现，与促旋酶不同，Topo IV结合时既不会将DNA限制在正超螺旋中，也不会保护150个碱基对的DNA区域不被微球菌核酸酶消化。与此一致的是，DNase I足迹实验表明，Topo IV保护了一个大约以拓扑异构酶诱导的切割位点为中心的34个碱基对的区域。此外，Topo IV优先结合超螺旋DNA而非松弛的DNA。因此，Topo IV的DNA结合特性更类似于II型真核酶，而非其原核伙伴的特性。
