Pedersen Ida Benedikte, Helgesen Emily, Flåtten Ingvild, Fossum-Raunehaug Solveig, Skarstad Kirsten
Department of Molecular Cell Biology and Department of Microbiology, Oslo University Hospital, P.O. Box 4950, 0424 Oslo, Norway.
School of Pharmacy, Faculty of Mathematics and Natural Sciences, University of Oslo, P.O. Box 4950, 0424 Oslo, Norway.
Nucleic Acids Res. 2017 Jun 20;45(11):6471-6485. doi: 10.1093/nar/gkx263.
The SeqA protein binds hemi-methylated GATC sites and forms structures that sequester newly replicated origins and trail the replication forks. Cells that lack SeqA display signs of replication fork disintegration. The broken forks could arise because of over-initiation (the launching of too many forks) or lack of dynamic SeqA structures trailing the forks. To confirm one or both of these possible mechanisms, we compared two seqA mutants with the oriCm3 mutant. The oriCm3 mutant over-initiates because of a lack of origin sequestration but has wild-type SeqA protein. Cells with nonfunctional SeqA, but not oriCm3 mutant cells, had problems with replication elongation, were highly dependent on homologous recombination, and exhibited extensive chromosome fragmentation. The results indicate that replication forks frequently break in the absence of SeqA function and that the broken forks are rescued by homologous recombination. We suggest that SeqA may act in two ways to stabilize replication forks: (i) by enabling vital replication fork repair and restarting reactions and (ii) by preventing replication fork rear-end collisions.
SeqA蛋白结合半甲基化的GATC位点并形成结构，这些结构会隔离新复制的起始点并跟随复制叉移动。缺乏SeqA的细胞表现出复制叉解体的迹象。断裂的复制叉可能是由于过度起始（启动了过多的复制叉）或缺乏跟随复制叉移动的动态SeqA结构而产生的。为了证实这两种可能机制中的一种或两种，我们将两个seqA突变体与oriCm3突变体进行了比较。oriCm3突变体由于缺乏起始点隔离而过度起始，但具有野生型SeqA蛋白。缺乏功能性SeqA的细胞，而不是oriCm3突变体细胞，在复制延伸方面存在问题，高度依赖同源重组，并表现出广泛的染色体片段化。结果表明，在缺乏SeqA功能的情况下，复制叉经常断裂，并且断裂的复制叉通过同源重组得以挽救。我们认为SeqA可能通过两种方式来稳定复制叉：（i）通过促进重要的复制叉修复和重新启动反应，以及（ii）通过防止复制叉后端碰撞。
