DYNAMIC LOCALIZATION OF PROTEINS AND CHROMOSOME IN BACTERIA

细菌中蛋白质和染色体的动态定位

基本信息

批准号：
11694221
负责人：
HIRAGA Sota
金额：
$ 10.25万
依托单位：
KUMAMOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11694221/
关键词：
DNA methyltransferase SeqA protein MukB protein replication fork DNA sliding clump sister chromosome cohesion DNAメチラーゼ SeqA MukB 染色体分配 MutS 複製装置 DNAメチレーション複製起点複製開始 DnaA Muts ミスマッチ修復

项目摘要

SeqA protein binds preferentially hemimethylated GATC sequences, and regulates negatively initiation of chromosome replication in E. coli. We demonstrated that purified SeqA inhibits binding of initiation protein DnaA to hemimethylated oriC DNA. In newborn cells, two MukB-GFP fluorescent foci are located at 1/4 and 3/4 cellular positions and one SeqA focus is located at the mid-cell position. Subsequently, a SeqA focus is divided into two and those migrate to the cell quarter positions. Beta-subunit (DnaN, DNA sliding clump) of DNA polymerase III holoenzyme is localized in cytosolic spaces at the cell poles in non-replicating cells. However, when chromosome replication is initiated, the subunit is recruited into nucleoid and forms a cluster at mid-cell. A beta-subunit cluster is separated into two that are located at the cell quarter positions, when two SeqA clusters are located at the cell quarter positions. These results support our 'translocating replication factories' model, in which paired replication apparatuses that act for bi-directional replication are first located closely with each other at mid-cell, and then migrate to the cell quarter positions. We found that sister chromosomes are cohesive with each other after replication and the cohesion is released in late period of 41 chromosome replication. Cohesion of sister chromosomes was not observed in MukB null mutant cells, indicating that MukB participates in sister chromosome cohesion. We analyzed localization of nascent DNA segments that were pulse-labeled with 5-bromodeoxyuridine and found that MukB is essential for cohesion of two replication forks and also for proper localization of forks.

SeqA 蛋白优先结合半甲基化的 GATC 序列，并负向调节大肠杆菌中染色体复制的起始。我们证明纯化的 SeqA 抑制起始蛋白 DnaA 与半甲基化 oriC DNA 的结合。在新生细胞中，两个 MukB-GFP 荧光焦点位于 1/4 和 3/4 细胞位置，一个 SeqA 焦点位于细胞中部位置。随后，SeqA 焦点被分成两部分，并迁移到细胞四分之一位置。 DNA聚合酶III全酶的β亚基（DnaN，DNA滑动团块）位于非复制细胞的细胞极的胞质空间中。然而，当染色体复制开始时，亚基被募集到核中并在细胞中部形成簇。当两个 SeqA 簇位于细胞四分之一位置时，β 亚基簇被分成位于细胞四分之一位置的两个。这些结果支持我们的“易位复制工厂”模型，其中用于双向复制的成对复制装置首先在细胞中部彼此靠近定位，然后迁移到细胞四分之一位置。我们发现姐妹染色体在复制后相互粘连，并且在41号染色体复制后期这种粘连被释放。在 MukB 无效突变细胞中未观察到姐妹染色体的凝聚，表明 MukB 参与姐妹染色体的凝聚。我们分析了用 5-溴脱氧尿苷脉冲标记的新生 DNA 片段的定位，发现 MukB 对于两个复制叉的凝聚以及叉的正确定位至关重要。