Single-molecule visualization of fast polymerase turnover in the bacterial replisome
Single-molecule visualization of fast polymerase turnover in the bacterial replisome
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DOI：
10.7554/elife.23932
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发表时间：
2017-04-22
期刊：
影响因子：
7.7
通讯作者：
van Oijen, Antoine M.
中科院分区：
文献类型：
--
作者：
Lewis, Jacob S.;Spenkelink, Lisanne M.;van Oijen, Antoine M.
大肠杆菌DNA复制机制已被用作揭示设计规则的路线图，使DNA复制具有高效率和保真度。虽然复制DNA Pol III的酶活性已被很好地理解，但其在复制体内的动力学尚不清楚。在这里，我们测试了公认的观点，即Pol III全酶在复制体内保持稳定的关联。我们使用荧光标记的聚合酶进行体外单分子分析，以证明Pol III*复合物（缺乏β 2滑动夹的全酶）在DNA复制过程中快速交换。然而，在溶液中没有Pol III*时，复制体具有高度的抗稀释性。我们进一步在含有标记钳装载器和聚合酶的活细胞中显示了类似的交换。这些观察结果表明，浓度依赖的交换机制提供了稳定性和可塑性之间的平衡，促进了依赖于其在环境中的可用性的复制体成分的替换。
The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here, we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the beta 2 sliding clamp), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled clamp loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment.