Measuring motion on DNA by the type I restriction endonuclease EcoR1241 using triplex displacement
Measuring motion on DNA by the type I restriction endonuclease EcoR1241 using triplex displacement
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DOI：
10.1093/emboj/19.9.2094
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发表时间：
2000-05-02
期刊：
影响因子：
11.4
通讯作者：
Szczelkun, MD
中科院分区：
文献类型：
--
作者：
Firman, K;Szczelkun, MD
I 型限制酶 EcoR124I 在依赖于 ATP 水解的广泛线性易位后切割 DNA。利用 DNA 三元组的蛋白质定向置换，我们确定了一维运动的动力学，而无需测量 DNA 或 ATP 水解。三链体是在来自 EcoR124I 位点 4370 bp 的线性 DNA 上特异性预形成的，然后与核酸内切酶一起孵育。添加 ATP 后，在三链体形成寡核苷酸以指数动力学取代之前观察到明显的滞后期。 I型和三联体位点之间的距离缩短，滞后时间减少，同时置换反应保持指数形式，这表明持续的DNA易位，随后与三联体和寡核苷酸置换发生碰撞。滞后持续时间和位点间距离之间的线性关系给出了 20°C 下 400 +/- 32 bp/s 的易位速度。此外，该数据只能通过高方向易位来解释。仅具有负责运动的两个 HsdR 亚基之一的核酸内切酶仍然可以催化易位。该反应进行性较差。但只要 DNA 被释放，就可以向任一方向“重置”。
The type I restriction enzyme EcoR124I cleaves DNA following extensive linear translocation dependent upon ATP hydrolysis. Using protein-directed displacement of a DNA triples, we have determined the kinetics of one-dimensional motion without the necessity of measuring DNA or ATP hydrolysis. The triplex was pre-formed specifically on linear DNA, 4370 bp from an EcoR124I site, and then incubated with endonuclease, Upon ATP addition, a distinct lag phase was observed before the triplex-forming oligonucleotide was displaced with exponential kinetics. iis the distance between type I and triples sites was shortened, the lag time decreased whilst the displacement reaction remained exponential, This is indicative of processive DNA translocation followed by collision with the tripler and oligonucleotide displacement. A linear relationship between lag duration and intersite distance gives a translocation velocity of 400 +/- 32 bp/s at 20 degrees C, Furthermore, the data can only be explained by hi-directional translocation, An endonuclease with only one of the two HsdR subunits responsible for motion could still catalyse translocation. The reaction is less processive. but can 'reset' in either direction whenever the DNA is released.