Single-molecule visualization of stalled replication-fork rescue by the Escherichia coli Rep helicase.
Single-molecule visualization of stalled replication-fork rescue by the Escherichia coli Rep helicase.
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DOI：
10.1093/nar/gkad186
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发表时间：
2023-04-24
影响因子：
14.9
通讯作者：
中科院分区：
文献类型：
--
作者：
当模板DNA被许多DNA结合蛋白结合时，就会发生基因组复制。这些蛋白质中的每一种都是复制分叉的潜在障碍，并可能对细胞产生有害影响。在大肠杆菌中，这些障碍被辅助解旋酶Rep取代，这是一种与复制体相互作用的DNA转位酶和解旋酶。与复制和代表清除障碍的协调背后的机制细节仍然知之甚少。通过对单个大肠杆菌复制体产生的DNA的实时荧光成像和荧光标记的Rep的同时可视化，我们发现Rep不断地监视延长的复制体。我们发现，Rep与复制体的这种关联是随机的，并且与叉子是否停滞无关。此外，当单个Rep分子从模板DNA中移除模型蛋白质障碍dCas9时，我们通过直接成像来可视化对停滞不前的复制叉子的有效救援。利用不同DNA结合稳定性的障碍，我们得出结论，合成的继续是停滞不前的复制拯救的限速步骤。
Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli, these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by individual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging individual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that continuation of synthesis is the rate-limiting step of stalled replication rescue.
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影响因子：
16
作者：
Guy, Colin P.;Atkinson, John;Gupta, Milind K.;Mahdi, Akeel A.;Gwynn, Emma J.;Rudolph, Christian J.;Moon, Peter B.;van Knippenberg, Ingeborg C.;Cadman, Chris J.;Dillingham, Mark S.;Lloyd, Robert G.;McGlynn, Peter
通讯作者：
McGlynn, Peter
影响因子：
11.4
作者：
Bidnenko, V;Ehrlich, SD;Michel, B
通讯作者：
Michel, B
影响因子：
16
作者：
Duderstadt, Karl E.;Geertsema, Hylkje J.;van Oijen, Antoine M.
通讯作者：
van Oijen, Antoine M.
影响因子：
5.6
作者：
Cheng, W;Hsieh, J;Lohman, TM
通讯作者：
Lohman, TM
DOI：
10.1073/pnas.242479399
发表时间：
2002-12-10
影响因子：
11.1
作者：
Cheng, W;Brendza, KM;Lohman, TM
通讯作者：
Lohman, TM