Real-time observation of a single DNA digestion by λ exonuclease under a fluorescence microscope field
Real-time observation of a single DNA digestion by λ exonuclease under a fluorescence microscope field
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DOI：
10.1093/nar/29.16.e79
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发表时间：
2001-08-15
影响因子：
14.9
通讯作者：
Mizuno, A
中科院分区：
文献类型：
--
作者：
Matsuura, S;Komatsu, J;Mizuno, A
荧光显微镜技术已经发展到可视化的行为个别DNA和蛋白质分子。对单个DNA分子的实时直接观察可用于研究DNA-蛋白质相互作用的动力学，例如λ核酸外切酶的DNA消化反应。在常规方法中，不可能分析DNA上单个λ核酸外切酶分子的动力学，因为它们只能观察许多核酸外切酶分子的平均行为。另一方面，对单个分子的观察可以揭示单个核酸外切酶分子的持续合成能力和结合速率。为了评估λ核酸外切酶的动力学，将具有一个生物素化末端的染色λ DNA分子固定在抗生物素蛋白包被的盖玻片上，并使用直流电场拉直。通过λ核酸外切酶消化拉直的DNA分子的显微镜观察显示，DNA消化速率类似于1000个碱基/秒，并且还证明了高的持续合成能力。
A fluorescence microscopy technique has been developed to visualize the behavior of individual DNA and protein molecules. Real-time direct observation of a single DNA molecule can be used to investigate the dynamics of DNA-protein interactions, such as the DNA digestion reaction by lambda exonuclease. In conventional methods it is impossible to analyze the dynamics of an individual lambda exonuclease molecule on a DNA because they can only observe the average behavior of a number of exonuclease molecules. Observation of a single molecule, on the other hand, can reveal processivity and binding rate of an individual exonuclease molecule. To evaluate the dynamics of lambda exonuclease, a stained lambda DNA molecule with one biotinylated terminal was fixed on an avidin-coated coverslip and straightened using a d.c. electric field. Microscopic observation of digestion of a straightened DNA molecule by lambda exonuclease revealed that the DNA digestion rate was similar to 1000 bases/s and also demonstrated high processivity.