Replication fork inhibition in seqA mutants of Escherichia coli triggers replication fork breakage.
Replication fork inhibition in seqA mutants of Escherichia coli triggers replication fork breakage.
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DOI：
10.1111/mmi.12638
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发表时间：
2014-07
影响因子：
3.6
通讯作者：
Kuzminov A
中科院分区：
文献类型：
--
作者：
Rotman E;Khan SR;Kouzminova E;Kuzminov A
SeqA蛋白负调控E.大肠杆菌，并提出组织成熟和分离的新复制的DNA。seqA突变体遭受染色体片段化;由于这种片段化归因于有缺陷的分离或类核压实，因此预期会出现两端断裂。相反，我们表明，在SeqA的情况下，染色体大多遭受单端DNA断裂，这表明复制叉的解体。我们进一步表明，复制叉是出乎意料的慢seqA突变体。从对齐的染色体的起点和末端复制的定量动力学不仅证实了seqA突变体中的起点过度起始，而且还揭示了末端复制不足，表明复制叉的抑制。在seqA突变体中的染色体片段化的前/后标记研究表明涉及单个叉的事件，而不是来自连续轮的成对叉彼此后端。我们认为，在缺乏SeqA的情况下，姐妹染色单体凝聚力的“安全间隔区”是不稳定的，完全消失，如果复制叉被抑制，导致分离叉运行到受抑制的复制叉和抢购它在单链DNA区域。
SeqA protein negatively regulates replication initiation in E. coli and is also proposed to organize maturation and segregation of the newly-replicated DNA. The seqA mutants suffer from chromosomal fragmentation; since this fragmentation is attributed to defective segregation or nucleoid compaction, two-ended breaks are expected. Instead, we show that, in SeqA’s absence, chromosomes mostly suffer one-ended DNA breaks, indicating disintegration of replication forks. We further show that replication forks are unexpectedly slow in seqA mutants. Quantitative kinetics of origin and terminus replication from aligned chromosomes not only confirm origin overinitiation in seqA mutants, but also reveal terminus underreplication, indicating inhibition of replication forks. Pre/post-labeling studies of the chromosomal fragmentation in seqA mutants suggest events involving single forks, rather than pairs of forks from consecutive rounds rear-ending into each other. We suggest that, in the absence of SeqA, the sister-chromatid cohesion “safety spacer” is destabilized and completely disappears if the replication fork is inhibited, leading to segregation fork running into the inhibited replication fork and snapping it at single-stranded DNA regions.
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