The Effect of Physical Form of DNA on ExonucleaseIII Activity Revealed by Single-molecule Observations
The Effect of Physical Form of DNA on ExonucleaseIII Activity Revealed by Single-molecule Observations
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DOI：
10.1007/s10895-008-0376-4
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发表时间：
2009-01-01
影响因子：
2.7
通讯作者：
Mizuno, Akira
中科院分区：
文献类型：
--
作者：
Kurita, Hirofumi;Torii, Ken;Mizuno, Akira
单分子研究揭示了通常隐藏在整体实验和时间平均中的分子行为。单分子测量可以控制单个DNA分子的物理形态，是获得DNA张力与酶活性相关性新知识的有力手段。在这里，我们研究了DNA的物理形式对核酸外切酶III（ExoIII）反应的影响。ExoIII具有双链DNA特异性3 '-> 5'核酸外切酶活性，并且消化是分布性的。我们观察到ExoIII从游离端消化单个拉伸的DNA分子。连续拍摄的照片表明，消化的DNA分子随反应时间线性缩短。我们还通过暂停缓冲液流进行了随机卷曲形式下的单分子观察。从两个单分子实验中获得的消化速率表明，拉伸条件下的消化速率是随机卷曲条件下的两倍。通过单分子观察清楚地证明了DNA的物理形式和ExoIII的消化速率之间的相关性。
Single-molecule studies have revealed molecular behaviors usually hidden in the ensemble and time averaging of bulk experiments. Single-molecule measurement that can control physical form of individual DNA molecules is a powerful method to obtain new knowledge about correlation between DNA-tension and enzyme activity. Here we study the effect of physical form of DNA on exonucleaseIII (ExoIII) reaction. ExoIII has a double-stranded DNA specific 3'-> 5' exonuclease activity and the digestion is distributive. We observed the ExoIII digestion of individual stretched DNA molecules from the free ends. The sequentially captured photographs demonstrated that the digested DNA molecule linearly shortened with the reaction time. We also carried out the single-molecule observation under random coiled form by pausing the buffer flow. The digestion rates obtained from both single-molecule experiments showed that the digestion rate under the stretched condition was two times higher than the random coiled condition. The correlation between physical form of DNA and digestion rate of ExoIII was clearly demonstrated by single-molecule observations.