Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo
Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo
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DOI：
10.1038/sj.emboj.7601155
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发表时间：
2006-06-07
期刊：
影响因子：
11.4
通讯作者：
Sherratt, David J.
中科院分区：
文献类型：
--
作者：
Possoz, Christophe;Filipe, Sergio R.;Sherratt, David J.
我们报告了一种有效的、可控的、特定部位的复制障碍，它阻止了细胞的增殖，但可以快速有效地逆转，导致生存能力的恢复。在10kb的操纵子DNA结合位点的抑制子结合阵列的前500bp内，两极的大肠杆菌复制叉子在体内停滞不前。抑制物结合的受控释放导致被阻断的复制分叉快速重新启动，而不参与同源重组。对分叉停滞和重新启动的细胞学跟踪表明，复制体相关的SSB蛋白在较长时间内仍与被阻断的分叉相关，并且与被阻断的操作者阵列相关的荧光焦点在重新启动后立即发生复制，从而表明阵列区域缺乏姐妹凝聚力。位于ORIC或DIF位点附近的路障并未阻止染色体其余部分的复制和分离。
We report an efficient, controllable, site-specific replication roadblock that blocks cell proliferation, but which can be rapidly and efficiently reversed, leading to recovery of viability. Escherichia coli replication forks of both polarities stalled in vivo within the first 500 bp of a 10 kb repressor-bound array of operator DNA-binding sites. Controlled release of repressor binding led to rapid restart of the blocked replication fork without the participation of homologous recombination. Cytological tracking of fork stalling and restart showed that the replisome-associated SSB protein remains associated with the blocked fork for extended periods and that duplication of the fluorescent foci associated with the blocked operator array occurs immediately after restart, thereby demonstrating a lack of sister cohesion in the region of the array. Roadblocks positioned near oriC or the dif site did not prevent replication and segregation of the rest of the chromosome.