Nucleic Acids Research Advance Access originally published online on June 10, 2008
Nucleic Acids Research 2008 36(12):4118-4127; doi:10.1093/nar/gkn376
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Nucleic Acids Research, 2008, Vol. 36, No. 12 4118-4127
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Sliding and jumping of single EcoRV restriction enzymes on non-cognate DNA
1Laboratoire Kastler Brossel, ENS, UPMC-Paris 6, CNRS UMR 8552, 24 rue Lhomond, 2Laboratoire de Physique Théorique de la Matière Condensée, CNRS UMR 7600, Université Pierre et Marie Curie-Paris 6, 4 place Jussieu, 3Muséum National d’Histoire Naturelle, INSERM U565, CNRS UMR 8646, 43 rue Cuvier, F-75005 Paris, France and 4Justus-Liebig-Universität Gießen, Institut für Biochemie, FB 8, Heinrich-Buff-Ring 58, D-32392 Gießen, Germany
*To whom correspondence should be addressed. Tel: +33 144 323 380; Fax: +33 144 323 434; Email: pierre.desbiolles{at}lkb.ens.fr
Correspondence may also be addressed to Andreas Biebricher. Tel: +33 144 323 455; Fax: +33 144 323 434; Email: biebrich{at}lkb.ens.fr
Received February 26, 2008. Revised May 27, 2008. Accepted May 28, 2008.
The restriction endonuclease EcoRV can rapidly locate a short recognition site within long non-cognate DNA using ‘facilitated diffusion’. This process has long been attributed to a sliding mechanism, in which the enzyme first binds to the DNA via nonspecific interaction and then moves along the DNA by 1D diffusion. Recent studies, however, provided evidence that 3D translocations (hopping/jumping) also help EcoRV to locate its target site. Here we report the first direct observation of sliding and jumping of individual EcoRV molecules along nonspecific DNA. Using fluorescence microscopy, we could distinguish between a slow 1D diffusion of the enzyme and a fast translocation mechanism that was demonstrated to stem from 3D jumps. Salt effects on both sliding and jumping were investigated, and we developed numerical simulations to account for both the jump frequency and the jump length distribution. We deduced from our study the 1D diffusion coefficient of EcoRV, and we estimated the number of jumps occurring during an interaction event with nonspecific DNA. Our results substantiate that sliding alternates with hopping/jumping during the facilitated diffusion of EcoRV and, furthermore, set up a framework for the investigation of target site location by other DNA-binding proteins.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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