Dynamics of Site Switching in DNA Polymerase
Dynamics of Site Switching in DNA Polymerase
复制标题
DNA 聚合酶位点转换的动力学
DOI：
10.1016/j.bpj.2012.11.2044
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发表时间：
2013
影响因子：
3.4
通讯作者：
D. Millar
中科院分区：
文献类型：
--
作者：
Rajan Lamichhane;S. Berezhna;Edwin Van;der Schans;D. Millar
DNA聚合酶通过催化脱氧核苷三磷酸（dNTP）底物在生长的DNA引物链的3 ′端上的模板定向聚合来复制DNA。许多DNA聚合酶还具有单独的3 ′-5 ′核酸外切酶活性，用于从新生DNA中去除错误掺入的核苷酸（校对）。聚合酶（pol）和核酸外切酶（exo）活性在不同的酶结构域中在空间上分离，这表明必须存在将生长的引物末端从一个位点转移到另一个位点的机制。在这里，我们报告了一个单分子Förster共振能量转移（smFRET）系统，直接监测DNA聚合酶I Klenow片段（Pol I KF）的pol和exo位点之间的DNA底物的运动。在单个聚合酶和DNA分子之间的相遇过程中记录的FRET轨迹揭示，DNA可以在两个方向上在pol和exo位点之间通道，同时保持与酶结合（分子内转移）。此外，很明显，DNA也可以从一个位点解离并在另一个位点重新结合（分子间转移）。通过对含有末端G·G错配的模型引物/模板的停留时间分析，确定了每种途径的速率常数，揭示了分子内转移是两种途径中较快的。令人惊讶的是，当dNTP底物也存在于溶液中时，错配的引物末端更频繁地访问外切位点，这有望增强校对。这些结果解释了Pol I KF的单独的pol和exo活动如何在物理上协调以实现有效的校对。结果还表明，核苷酸在聚合酶功能过程中的作用扩大。
DNA polymerases replicate DNA by catalyzing the template-directed polymerization of deoxynucleoside triphosphate (dNTP) substrates onto the 3′ end of a growing DNA primer strand. Many DNA polymerases also possess a separate 3′-5′ exonuclease activity that is used to remove misincorporated nucleotides from the nascent DNA (proofreading). The polymerase (pol) and exonuclease (exo) activities are spatially separated in different enzyme domains, indicating that a mechanism must exist to transfer the growing primer terminus from one site to the other. Here we report a single-molecule Förster resonance energy transfer (smFRET) system that directly monitors the movement of a DNA substrate between the pol and exo sites of DNA polymerase I Klenow fragment (Pol I KF). FRET trajectories recorded during the encounter between single polymerase and DNA molecules reveal that DNA can channel between the pol and exo sites in both directions while remaining bound to the enzyme (intramolecular transfer). In addition, it is evident that DNA can also dissociate from one site and rebind at the other (intermolecular transfer). Rate constants for each pathway have been determined by dwell-time analysis for a model primer/template containing a terminal G• G mispair, revealing that intramolecular transfer is the faster of the two pathways. Surprisingly, the mispaired primer terminus accesses the exo site more frequently when dNTP substrates are also present in solution, which is expected to enhance proofreading. These results explain how the separate pol and exo activities of Pol I KF are physically coordinated to achieve efficient proofreading. The results also suggest an expanded role for nucleotides during polymerase function.