Dynamics of Proofreading by the E. coli Pol III Replicase
Dynamics of Proofreading by the E. coli Pol III Replicase
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DOI：
10.1016/j.chembiol.2017.09.008
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发表时间：
2018-01-01
影响因子：
8.6
通讯作者：
Lee, Jong-Bong
中科院分区：
文献类型：
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作者：
Park, Jonghyun;Jergic, Slobodan;Lee, Jong-Bong
大肠杆菌DNA聚合酶III（Pol III）的α-β-theta核心与β（2）滑动夹结合，以促进DNA合成并去除错误掺入的核苷酸。α亚基是聚合酶，而β是3'至5'校对核酸外切酶。相反，聚合酶活性的聚合酶III，动态功能的校对知之甚少。我们使用单分子测定来确定β（2）相关的Pol III核心的切除率和持续合成能力，并观察到这两种性质通过突变加强β（2）和β（2）之间的相互作用而增强。因此，ε-β（2）接触在合成和校对模式中都保持。值得注意的是，单分子实时荧光成像揭示了引物-模板DNA在聚合酶和校对位点之间转移的动力学，表明它不涉及破坏β-内酰胺酶和β-内酰胺酶之间的物理相互作用。
The alpha epsilon theta core of Escherichia coli DNA polymerase III (Pol III) associates with the beta(2) sliding clamp to processively synthesize DNA and remove misincorporated nucleotides. The alpha subunit is the polymerase while epsilon is the 3' to 5' proofreading exonuclease. In contrast to the polymerase activity of Pol III, dynamic features of proofreading are poorly understood. We used single-molecule assays to determine the excision rate and processivity of the beta(2)-associated Pol III core, and observed that both properties are enhanced by mutational strengthening of the interaction between epsilon and beta(2). Thus, the epsilon-beta(2) contact is maintained in both the synthesis and proofreading modes. Remarkably, single-molecule real-time fluorescence imaging revealed the dynamics of transfer of primer-template DNA between the polymerase and proofreading sites, showing that it does not involve breaking of the physical interaction between epsilon and beta(2) .