Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes
Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes
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DOI：
10.1016/j.molcel.2016.10.028
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发表时间：
2016-12-15
期刊：
影响因子：
16
通讯作者：
van Oijen, Antoine M.
中科院分区：
文献类型：
--
作者：
Duderstadt, Karl E.;Geertsema, Hylkje J.;van Oijen, Antoine M.
负责DNA复制的分子机制，即复制体，必须有效地协调DNA的解绕与引物和合成，以完成两条链的复制。由于DNA的反平行特性，前导链是连续复制的，而后导链是通过引物、DNA环和冈崎片段合成的重复循环产生的。在这里，我们报告了一种多维单分子方法，通过同时监测环生长和导链合成的动力学，来可视化噬菌体T7复制体中的这种协调。我们发现滞后链上的环主要发生在引物期间，并且很少支持随后的冈崎片段合成。荧光成像显示聚合酶仍然与复制叉后面的滞后链结合，与冈崎片段合成一致，独立于复制复合体。单个复制体在启动过程中显示出循环和暂停，这调和了引物合成调节的不同模型，并揭示了复制体操作中潜在的可塑性。
The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multi-dimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.