期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 104, 期 31, 页码 12755-12760出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0700483104
关键词
imaging; nucleic acid; restriction-modification enzyme; scanning-probe
资金
- Biotechnology and Biological Sciences Research Council [BBS/B/1065X] Funding Source: Medline
- Biotechnology and Biological Sciences Research Council [BBS/B/1065X] Funding Source: researchfish
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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据