Huang He, Song Cheng-Cheng, Yang Zhi-Liang, Dong Yan, Hu Yao-Zhong, Gao Feng
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University Tianjin, China ; Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University Tianjin, China ; Collaborative Innovation Center of Chemical Science and Engineering Tianjin, China.
Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University Tianjin, China ; Collaborative Innovation Center of Chemical Science and Engineering Tianjin, China ; Department of Physics, Tianjin University Tianjin, China.
Front Microbiol. 2015 Dec 10;6:1370. doi: 10.3389/fmicb.2015.01370. eCollection 2015.
Based on the complete genome of Cyanothece ATCC 51142, the oriCs of both the circular and linear chromosomes in Cyanothece ATCC 51142 have been predicted by utilizing a web-based system Ori-Finder. Here, we provide experimental support for the results of Ori-Finder to identify the replication origins of Cyanothece ATCC 51142 and their interactions with the initiator protein, DnaA. The two replication origins are composed of three characteristically arranged DnaA boxes and an AT-rich stretch, and the oriC in the circular chromosome is followed by the dnaN gene. The dnaA gene is located downstream of the origin of the circular chromosome and it expresses a typical DnaA protein that is divided into four domains (I, II, III, IV), as with other members of the DnaA protein family. We purify DnaA (IV) and characterize the interaction of the purified protein with the replication origins, so as to offer experimental support for the prediction. The results of the electrophoretic mobility shift assay and DNase I footprint assay demonstrate that the C-terminal domain of the DnaA protein from Cyanothece ATCC 51142 specifically binds the oriCs of both the circular and linear chromosomes, and the DNase I footprint assay demonstrates that DnaA (IV) exhibits hypersensitive affinity with DnaA boxes in both oriCs.
基于蓝细菌ATCC 51142的全基因组，利用基于网络的Ori-Finder系统预测了蓝细菌ATCC 51142中环状和线性染色体的oriC。在此，我们为Ori-Finder的结果提供实验支持，以鉴定蓝细菌ATCC 51142的复制起点及其与起始蛋白DnaA的相互作用。这两个复制起点由三个特征性排列的DnaA框和一个富含AT的区域组成，环状染色体中的oriC后面跟着dnaN基因。dnaA基因位于环状染色体起点的下游，它表达一种典型的DnaA蛋白，该蛋白分为四个结构域（I、II、III、IV），与DnaA蛋白家族的其他成员一样。我们纯化了DnaA（IV）并表征了纯化蛋白与复制起点的相互作用，从而为预测提供实验支持。电泳迁移率变动分析和DNase I足迹分析的结果表明，来自蓝细菌ATCC 51142的DnaA蛋白的C末端结构域特异性结合环状和线性染色体的oriC，并且DNase I足迹分析表明DnaA（IV）对两个oriC中的DnaA框表现出超敏亲和力。
