Li Zhiyu, Hiasa Hiroshi, DiGate Russell
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MA, USA.
Nucleic Acids Res. 2005 Sep 28;33(17):5415-25. doi: 10.1093/nar/gki846. Print 2005.
The Bacillus cereus genome possesses three type IA topoisomerase genes. These genes, encoding DNA topoisomerase I and IIIalpha (bcTopo I, bcTopo IIIalpha), have been cloned into T7 RNA polymerase-regulated plasmid expression vectors and the enzymes have been overexpressed, purified and characterized. The proteins exhibit similar biochemical activity to their Escherichia coli counterparts, DNA topoisomerase I and III (ecTopo I, ecTopo III). bcTopo I is capable of efficiently relaxing negatively supercoiled DNA in the presence of Mg2+ but does not possess an efficient DNA decatenation activity. bcTopo IIIalpha is an active topoisomerase that is capable of relaxing supercoiled DNA at a broad range of Mg2+ concentrations; however, its DNA relaxation activity is not as efficient as that of bcTopo I. In addition, bcTopo III is a potent DNA decatenase that resolves oriC-based plasmid replication intermediates in vitro. Interestingly, bcTopo I and bcTopo IIIalpha are both able to compensate for the loss of ecTopo III in E.coli cells that lack ecTopo I. In contrast, ecTopo I cannot substitute for ecTopo III under these conditions.
蜡样芽孢杆菌基因组含有三个IA型拓扑异构酶基因。这些编码DNA拓扑异构酶I和IIIα（bcTopo I、bcTopo IIIα）的基因已被克隆到T7 RNA聚合酶调控的质粒表达载体中，并且这些酶已被过量表达、纯化和鉴定。这些蛋白质与其大肠杆菌对应物DNA拓扑异构酶I和III（ecTopo I、ecTopo III）表现出相似的生化活性。bcTopo I在Mg2+存在下能够有效地松弛负超螺旋DNA，但不具有有效的DNA解连环活性。bcTopo IIIα是一种活性拓扑异构酶，能够在广泛的Mg2+浓度范围内松弛超螺旋DNA；然而，其DNA松弛活性不如bcTopo I高效。此外，bcTopo III是一种强大的DNA解连环酶，能够在体外解析基于oriC的质粒复制中间体。有趣的是，bcTopo I和bcTopo IIIα都能够补偿缺乏ecTopo I的大肠杆菌细胞中ecTopo III的缺失。相反，在这些条件下ecTopo I不能替代ecTopo III。
