Distinct regions of the Escherichia coli ParC C-terminal domain are required for substrate discrimination by topoisomerase IV.
Distinct regions of the Escherichia coli ParC C-terminal domain are required for substrate discrimination by topoisomerase IV.
复制标题
拓扑异构酶 IV 区分底物需要大肠杆菌 ParC C 末端结构域的不同区域。
DOI：
10.1016/j.jmb.2013.04.033
复制
发表时间：
2013
影响因子：
5.6
通讯作者：
Berger,JamesM
中科院分区：
文献类型：
--
作者：
Vos,SeychelleM;Lee,Imsang;Berger,JamesM
IIA型DNA拓扑异构酶是利用ATP维持染色体超螺旋和去除姐妹染色体之间的连接的必需酶。在大肠杆菌中，IIA型拓扑异构酶topo IV迅速地从DNA中去除正超螺旋和索烃，但当面对负超螺旋底物时，速度明显较慢。topo IV区分正超螺旋DNA和负超螺旋DNA的能力需要其两个亚基之一ParC的C末端结构域（CTD）。为了确定ParC CTD如何有助于底物识别，我们鉴定了CTD表面上潜在的DNA相互作用残基，突变这些残基，并测试了它们对分离结构域的拓扑异构酶IV酶活性和DNA结合的影响。令人惊讶的是，ParC CTD的不同区域不等同地结合DNA，也不等同地促进拓扑异构体IV对不同类型的DNA底物的作用。此外，我们发现，CTD包含一个自动抑制元件，抑制活性的负超螺旋和连锁底物，以及一个独特的区域，有助于弯曲的DNA双链体，通过酶的溶核中心跟踪。我们的数据表明，CTD是必不可少的门和传输段DNA的适当参与，调和不同的模型来解释拓扑IV如何区分不同的DNA拓扑结构。
Type IIA DNA topoisomerases are essential enzymes that use ATP to maintain chromosome supercoiling and remove links between sister chromosomes. InEscherichia coli, the type IIA topoisomerase topo IV rapidly removes positive supercoils and catenanes from DNA but is significantly slower when confronted with negatively supercoiled substrates. The ability of topo IV to discriminate between positively and negatively supercoiled DNA requires the C-terminal domain (CTD) of one of its two subunits, ParC. To determine how the ParC CTD might assist with substrate discrimination, we identified potential DNA interacting residues on the surface of the CTD, mutated these residues, and tested their effect on both topo IV enzymatic activity and DNA binding by the isolated domain. Surprisingly, different regions of the ParC CTD do not bind DNA equivalently, nor contribute equally to the action of topo IV on different types of DNA substrates. Moreover, we find that the CTD contains an autorepressive element that inhibits activity on negatively supercoiled and catenated substrates, as well as a distinct region that aids in bending the DNA duplex that tracks through the enzyme's nucleolytic center. Our data demonstrate that the CTD is essential for proper engagement of both gate and transfer segment DNAs, reconciling different models to explain how topo IV discriminates between distinct DNAs topologies.