Real-time direct observation of single-molecule DNA hydrolysis by exonucleaseIII
Real-time direct observation of single-molecule DNA hydrolysis by exonucleaseIII
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DOI：
10.1080/07391102.2008.10507194
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发表时间：
2006-11
期刊：
影响因子：
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通讯作者：
H. Kurita;K. Inaishi;K. Torii;M. Urisu;M. Nakano;S. Katsura;A. Mizuno
中科院分区：
文献类型：
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作者：
H. Kurita;K. Inaishi;K. Torii;M. Urisu;M. Nakano;S. Katsura;A. Mizuno
利用荧光技术对单个分子进行真实的实时直接观察，不仅可以直接表征分子的平均行为，而且可以直接表征分子的分布和涨落，从而解释了许多生物现象，而传统的方法只能观察分子的平均行为。近年来，单分子分析的各种DNA-蛋白质相互作用，通过使用荧光技术进行。在这项研究中，观察到单分子DNA水解的外切核酸酶III，它具有3'和5'外切核酸酶活性。这种单分子观测方法基于光阱和两层层流，其中两层之间的混合可以忽略不计。首先，一个DNA-珠复合物被光学捕获在没有核酸外切酶III的层中。这种捕获允许DNA通过流动拉伸。通过将捕获的DNA-珠复合物移动到另一个含有核酸外切酶III的流动层来启动核酸外切酶III反应，然后在真实的时间内观察荧光染色的DNA的缩短。顺序捕获的照片表明，消化的DNA分子随反应时间线性缩短。测定了单分子实验的消化率，并与体外实验进行了比较。结果表明，单分子实验中核酸外切酶III的消化率比体外消化率高约20倍
Real time direct observation of single molecule using fluorescent technique has elucidated various biological phenomena, because not only average but also distributions and fluctuations of molecules can be characterized directly by this method, on the other hand, conventional methods can observe only average behaviors of molecules. In recent years, single-molecule analyses of various DNA-protein interactions by using florescent techniques were performed. In this study, single-molecule DNA hydrolysis by exonucleaseIII was observed, which has 3' rarr 5' exonuclease activity. This single-molecule observation method was based on optical trap and two-layer laminar flow, in which mixing between the two layers was negligible. First of all, one DNA-bead complex was optically trapped in a layer without exonucleaseIII. This trapping permitted stretching of DNA by flow. ExonucleaseIII reaction was initiated by moving the trapped DNA-bead complex to another flow layer containing exonucleaseIII, then shortening of fluorescently stained DNA was observed in real time. The sequentially captured photographs demonstrate that the digested DNA molecule linearly shortened with reaction time. The digestion rate from single-molecule experiment was determined and compared to in vitro experiment. As a result, digestion rate of exonucleaseIII from single-molecule experiment was ~20 times higher than in vitro digestion rate