Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution
Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution
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DOI：
10.1073/pnas.0908640106
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发表时间：
2009-12-15
影响因子：
11.1
通讯作者：
Rueda, David
中科院分区：
文献类型：
--
作者：
Christian, Thomas D.;Romano, Louis J.;Rueda, David
DNA聚合酶的催化机制涉及在核苷酸转移到生长的DNA链的3 '-羟基之前和之后的多个步骤。在这里，我们报告了一个单分子的方法来监测运动的E。大肠杆菌DNA聚合酶I（Klenow片段）在DNA合成过程中对DNA模板的单碱基对分辨率。当每个核苷酸被掺入时，单分子福斯特共振能量转移强度以离散步骤下降到与单核苷酸构象一致的值。嘌呤和嘧啶的掺入率相当。错配的引物/模板连接表现出与移动到核酸外切酶结构域中的引物一致的动力学，其用于确定与核酸外切酶和聚合酶位点结合的引物末端的分数。最有趣的是，我们观察到一个结构变化后，掺入一个正确配对的核苷酸，与聚合酶过去的预插入位点或构象变化的聚合酶的瞬时运动一致。这可能代表了DNA合成机制中以前未观察到的步骤，可能是校对过程的一部分。
The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3'-hydroxyl of the growing DNA chain. Here we report a single-molecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule Forster resonance energy transfer intensity drops in discrete steps to values consistent with single-nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer/template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change after the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the preinsertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.