Nozaki Shingo, Niki Hironori, Ogawa Tohru
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, Japan.
J Bacteriol. 2009 Aug;191(15):4807-14. doi: 10.1128/JB.00435-09. Epub 2009 Jun 5.
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 DeltadatA 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.
DnaA是一种在细菌中保守的复制起始蛋白。它在DNA复制起始过程中发挥核心作用。为了监测其在活的大肠杆菌细胞中的行为，该蛋白的一个非必需部分被荧光蛋白取代。这样的菌株生长正常，流式细胞术数据表明嵌合蛋白的起始活性没有实质性损失。起始蛋白分布在整个类核中。此外，大多数细胞表现出某些发出明亮荧光的明显焦点。这些焦点与复制起点（oriC）区域共定位，并且在跨越起始事件的期间最亮。在已经经历起始的细胞中，焦点富集在强度较低的区域。此外，在这个细胞周期阶段，很大一部分oriC区域没有共定位的DnaA增强型黄色荧光蛋白（EYFP）焦点。很难将起始蛋白滴定位点（datA）与oriC区域区分开来。然而，从以下观察结果表明datA参与起始控制：在缺失datA的细胞中，DnaA-EYFP在细胞周期中比野生型细胞更早地与oriC区域最大程度地共定位，并且oriC浓度增加。
