Capturing reaction paths and intermediates in Cre-loxP recombination using single-molecule fluorescence
Capturing reaction paths and intermediates in Cre-loxP recombination using single-molecule fluorescence
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使用单分子荧光捕获 Cre-loxP 重组中的反应路径和中间体
DOI：
10.1073/pnas.1211922109
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
A. Kapanidis
中科院分区：
文献类型：
--
作者：
Justin N. M. Pinkney;P. Zawadzki;Jarosław Mazuryk;L. Arciszewska;D. Sherratt;A. Kapanidis
位点特异性重组在微生物生物学中起着关键作用，并被广泛用于操纵高等生物的基因组。Cre是一种位点特异性重组酶，负责在细菌中建立和维持P1噬菌体基因组。在重组过程中，Cre在两个34 bp的DNA序列之间形成称为loxP的突触复合物，之后一对链交换形成霍利迪连接（HJ）中间体;然后HJ异构化允许第二对链交换，从而形成最终的重组产物。尽管在Cre-loxP系统上进行了大量的工作，但其许多机制仍然不清楚，主要是由于形成的复合物的瞬时性质和大多数生物化学工作所固有的系综平均。在这里，我们通过引入系留荧光团运动（TFM），一种通过单个荧光团的扩散自由度的报告来监测大规模DNA运动的方法来解决这些限制。我们结合联合收割机TFM与Förster共振能量转移（FRET），同时观察大规模和小规模的构象变化在单个DNA分子。使用TFM-FRET，我们观察到个别重组反应在真实的时间，并分析其动力学。主要通过交换DNA底物的“底链”来启动降解。在生产复合物中，我们使用FRET分布来推断HJ中间体的快速异构化，并且在该异构化之后发生限速步骤。我们还观察到两个nonproductive突触复合物，其中之一是结构上不同的晶体中的构象。重组后，产物突触复合物非常稳定，并且对随后的几轮重组是难治的。
Site-specific recombination plays key roles in microbe biology and is exploited extensively to manipulate the genomes of higher organisms. Cre is a well studied site-specific recombinase, responsible for establishment and maintenance of the P1 bacteriophage genome in bacteria. During recombination, Cre forms a synaptic complex between two 34-bp DNA sequences called loxP after which a pair of strand exchanges forms a Holliday junction (HJ) intermediate; HJ isomerization then allows a second pair of strand exchanges and thus formation of the final recombinant product. Despite extensive work on the Cre-loxP system, many of its mechanisms have remained unclear, mainly due to the transient nature of complexes formed and the ensemble averaging inherent to most biochemical work. Here, we address these limitations by introducing tethered fluorophore motion (TFM), a method that monitors large-scale DNA motions through reports of the diffusional freedom of a single fluorophore. We combine TFM with Förster resonance energy transfer (FRET) and simultaneously observe both large- and small-scale conformational changes within single DNA molecules. Using TFM–FRET, we observed individual recombination reactions in real time and analyzed their kinetics. Recombination was initiated predominantly by exchange of the “bottom-strands” of the DNA substrate. In productive complexes we used FRET distributions to infer rapid isomerization of the HJ intermediates and that a rate-limiting step occurs after this isomerization. We also observed two nonproductive synaptic complexes, one of which was structurally distinct from conformations in crystals. After recombination, the product synaptic complex was extremely stable and refractory to subsequent rounds of recombination.
影响因子：
3.4
作者：
Lee, NK;Kapanidis, AN;Weiss, S
通讯作者：
Weiss, S