Single-molecule analysis uncovers the difference between the kinetics of DNA decatenation by bacterial topoisomerases I and III.
Single-molecule analysis uncovers the difference between the kinetics of DNA decatenation by bacterial topoisomerases I and III.
复制标题
单分子分析揭示了细菌拓扑异构酶 I 和 III 的 DNA 串联动力学之间的差异。
DOI：
10.1093/nar/gku785
复制
发表时间：
2014
影响因子：
14.9
通讯作者：
Mondragón,Alfonso
中科院分区：
文献类型：
--
作者：
Terekhova,Ksenia;Marko,JohnF;Mondragón,Alfonso
大肠杆菌核糖体异构酶I和III可以使含有单链DNA区域或切口的双链DNA（dsDNA）分子解链，以及使负超螺旋DNA松弛。虽然这些蛋白质具有相同的作用机制和相似的结构，但它们参与不同的细胞过程。而拓扑异构酶III是一个更有效的decatenase比拓扑异构酶I，相反的是正确的DNA松弛。为了研究这两种原型IA型拓扑异构酶的机制的差异，我们研究了DNA decatenation在单分子水平上使用辫子的完整的dsDNA和缺口的dsDNA与凸起。我们发现，这两种蛋白质都不能使完整的DNA辫状结构降解。相比之下，这两种酶表现出强大的脱连环活性的DNA辫子与凸起。实验表明，这些拓扑异构酶解编织机制之间的主要差异在于解链循环之间的停顿。拓扑异构酶III的暂停时间越短，脱链率越高。此外，拓扑异构酶III显示出对DNA链的交叉角的强烈依赖性。这些实时观测揭示了去连环化机制的动力学特征，并有助于解释它们的活动之间的差异。
Escherichia colitopoisomerases I and III can decatenate double-stranded DNA (dsDNA) molecules containing single-stranded DNA regions or nicks as well as relax negatively supercoiled DNA. Although the proteins share a mechanism of action and have similar structures, they participate in different cellular processes. Whereas topoisomerase III is a more efficient decatenase than topoisomerase I, the opposite is true for DNA relaxation. In order to investigate the differences in the mechanism of these two prototypical type IA topoisomerases, we studied DNA decatenation at the single-molecule level using braids of intact dsDNA and nicked dsDNA with bulges. We found that neither protein decatenates an intact DNA braid. In contrast, both enzymes exhibited robust decatenation activity on DNA braids with a bulge. The experiments reveal that a main difference between the unbraiding mechanisms of these topoisomerases lies in the pauses between decatenation cycles. Shorter pauses for topoisomerase III result in a higher decatenation rate. In addition, topoisomerase III shows a strong dependence on the crossover angle of the DNA strands. These real-time observations reveal the kinetic characteristics of the decatenation mechanism and help explain the differences between their activities.