Identification of the Replication Origins from Cyanothece ATCC 51142 and Their Interactions with the DnaA Protein: From In Silico to In Vitro Studies.
Identification of the Replication Origins from Cyanothece ATCC 51142 and Their Interactions with the DnaA Protein: From In Silico to In Vitro Studies.
复制标题
Cyanothece ATCC 51142 复制起点的鉴定及其与 DnaA 蛋白的相互作用：从计算机研究到体外研究
DOI：
10.3389/fmicb.2015.01370
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发表时间：
2015
影响因子：
5.2
通讯作者：
Gao F
中科院分区：
文献类型：
--
作者：
Huang H;Song CC;Yang ZL;Dong Y;Hu YZ;Gao F
基于蓝杆藻ATCC 51142的全基因组，利用Ori-PCR网络系统对蓝杆藻ATCC 51142的环状和线状染色体的oriCs进行了预测。在这里，我们为Ori-Finder的结果提供了实验支持，以鉴定蓝丝菌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盒和一个富含AT的延伸段组成，环状染色体中的oriC后面是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，该蛋白被分为四个结构域（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DNA酶I足迹分析的结果表明，来自蓝杆藻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DNA酶I足迹分析表明，DnaA（IV）与两种oriC中的DnaA盒表现出超敏亲和力。
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.