Replication Initiator DnaA of Escherichia coli Changes Its Assembly Form on the Replication Origin during the Cell Cycle
Replication Initiator DnaA of Escherichia coli Changes Its Assembly Form on the Replication Origin during the Cell Cycle
复制标题
DOI：
10.1128/jb.00435-09
复制
发表时间：
2009-06
影响因子：
3.2
通讯作者：
Shingo Nozaki;H. Niki;T. Ogawa
中科院分区：
文献类型：
--
作者：
Shingo Nozaki;H. Niki;T. Ogawa
dna是一种在细菌中保守的复制启动蛋白。它在DNA复制的起始阶段起着核心作用。为了监测其在活的大肠杆菌细胞中的行为，蛋白质的非必需部分被荧光蛋白取代。这种菌株生长正常，流式细胞术数据表明嵌合蛋白没有实质性的引发剂活性损失。引发剂分布在整个类核上。此外，大多数细胞表现出某些独特的焦点，发出明亮的荧光。这些焦点与复制起始区（oriC）共定位，并且在起始事件期间最亮。在经历起始的细胞中，灶在强度较低的细胞中富集。此外，该细胞周期阶段的oriC区域中有很大一部分没有共定位的dna增强黄色荧光蛋白（EYFP）焦点。引发剂滴定位点（datA）与oriC区难以区分。然而，通过观察发现，在ΔdatA细胞中，与野生型细胞相比，dna - eyfp在细胞周期中更早地与oriC区域共定位，并且oriC浓度增加，这表明datA参与了起始控制。
ABSTRACT DnaA is a replication initiator protein that is conserved among bacteria. It plays a central role in the initiation of DNA replication. In order to monitor its behavior in living Escherichia coli cells, a nonessential portion of the protein was replaced by a fluorescent protein. Such a strain grew normally, and flow cytometry data suggested that the chimeric protein has no substantial loss of the initiator activity. The initiator was distributed all over the nucleoid. Furthermore, a majority of the cells exhibited certain distinct foci that emitted bright fluorescence. These foci colocalized with the replication origin (oriC) region and were brightest during the period spanning the initiation event. In cells that had undergone the initiation, the foci were enriched in less intense ones. In addition, a significant portion of the oriC regions at this cell cycle stage had no colocalized DnaA-enhanced yellow fluorescent protein (EYFP) focus point. It was difficult to distinguish the initiator titration locus (datA) from the oriC region. However, involvement of datA in the initiation control was suggested from the observation that, in ΔdatA cells, DnaA-EYFP maximally colocalized with the oriC region earlier in the cell cycle than it did in wild-type cells and oriC concentration was increased.