Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP151 is capable of DNA translocation and looping
Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP151 is capable of DNA translocation and looping
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DOI：
10.1073/pnas.0700483104
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发表时间：
2007-07-31
影响因子：
11.1
通讯作者：
Henderson, Robert M.
中科院分区：
文献类型：
--
作者：
Crampton, Neal;Yokokawa, Masatoshi;Henderson, Robert M.
许多DNA修饰酶的作用方式需要两个不相邻的DNA位点之间的通信。这些位点可以通过结合在两个位点的酶之间的扩散介导的偶然相互作用，或者通过位点结合的酶对插入DNA的主动易位而接触。EcoP 151是一种III型限制性内切酶，在切割DNA之前，它需要与两个相隔3,500 bp的识别位点相互作用。在这里，我们已经研究了EcoP 151的行为，使用一种新型的快速扫描原子力显微镜，它使用一个小型化的悬臂和扫描阶段，以减少悬臂的机械响应时间，并防止在高扫描速度的共振运动的发生。有了这台仪器，我们能够在流体下实现高达每秒10帧的扫描速率。改进的时间分辨率使我们能够以每秒1-3帧的扫描速率对EcoPl 51进行真实的成像。EcoP 151以ATIP依赖的方式易位DNA，速率为79 +/- 33 bp/s。也可以观察到超螺旋的积累，这是EcoP 151沿着DNA运动的结果。EcoP 151结合到其识别位点上，也可以与其他DNA位点进行非特异性接触，从而形成DNA环并缩短两个识别位点之间的距离。根据我们的研究结果，我们得出结论，EcoP 151使用两种不同的机制，两个识别位点之间的通信：扩散DNA环的形成和ATP酶驱动的易位的干预DNA轮廓。
Many DNA-modifying enzymes act in a manner that requires communication between two noncontiguous DNA sites. These sites can be brought into contact either by a diffusion-mediated chance interaction between enzymes bound at the two sites, or by active translocation of the intervening DNA by a site-bound enzyme. EcoP151, a type III restriction enzyme, needs to interact with two recognition sites separated by up to 3,500 bp before it can cleave DNA. Here, we have studied the behavior of EcoP151, using a novel fast-scan atomic force microscope, which uses a miniaturized cantilever and scan stage to reduce the mechanical response time of the cantilever and to prevent the onset of resonant motion at high scan speeds. With this instrument, we were able to achieve scan rates of up to 10 frames per s under fluid. The improved time resolution allowed us to image EcoPl51 in real time at scan rates of 1-3 frames per s. EcoP151 translocated DNA in an ATIP-dependent manner, at a rate of 79 +/- 33 bp/s. The accumulation of supercoiling, as a consequence of movement of EcoP151 along the DNA, could also be observed. EcoP151 bound to its recognition site was also seen to make nonspecific contacts with other DNA sites, thus forming DNA loops and reducing the distance between the two recognition sites. On the basis of our results, we conclude that EcoP151 uses two distinct mechanisms to communicate between two recognition sites: diffusive DNA loop formation and ATPase-driven translocation of the intervening DNA contour.