Visualizing helicases unwinding DNA at the single molecule level.
Visualizing helicases unwinding DNA at the single molecule level.
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DOI：
10.1093/nar/gkq173
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发表时间：
2010-07
影响因子：
14.9
通讯作者：
Molloy JE
中科院分区：
文献类型：
--
作者：
Fili N;Mashanov GI;Toseland CP;Batters C;Wallace MI;Yeeles JT;Dillingham MS;Webb MR;Molloy JE
DNA解旋酶是一种马达蛋白，利用ATP水解产生的自由能催化双链DNA解绕成单链DNA。单分子方法使我们能够解决这些酶如何沿着DNA前进的详细机制问题。在这里，已经开发出一种光学方法来实时跟踪多个DNA分子同时展开。这是通过使用全内反射荧光显微镜测量解旋酶单链DNA产物上荧光单链DNA结合蛋白的积累来实现的。通过固定DNA或解旋酶，局部荧光的增加提供了关于解绕速率和单个酶的处理能力的信息。此外，它还揭示了展开过程的细节，如暂停和活动的爆发。该分析的通用和通用性质使其适用于各种DNA解旋酶和DNA模板。该方法是用于研究DNA加工酶的单分子工具箱的重要补充。
DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes.
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影响因子：
4.8
作者：
Chedin, Frederic;Handa, Naofumi;Kowalczykowski, Stephen C.
通讯作者：
Kowalczykowski, Stephen C.
影响因子：
4.8
作者：
Mashanov, GI;Tacon, D;Molloy, JE
通讯作者：
Molloy, JE
影响因子：
2.9
作者：
Kunzelmann, Simone;Morris, Caroline;Webb, Martin R.
通讯作者：
Webb, Martin R.
DOI：
10.1073/pnas.87.21.8560
发表时间：
1990-11-01
影响因子：
11.1
作者：
NOIROT, P;BARGONETTI, J;NOVICK, RP
通讯作者：
NOVICK, RP
影响因子：
3.6
作者：
Petit, MA;Dervyn, E;Bruand, C
通讯作者：
Bruand, C