Sliding and jumping of single EcoRV restriction enzymes on non-cognate DNA
Open Access
- 10 June 2008
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 36 (12), 4118-4127
- https://doi.org/10.1093/nar/gkn376
Abstract
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.Keywords
This publication has 52 references indexed in Scilit:
- Dynamic Basis for One-Dimensional DNA Scanning by the Mismatch Repair Complex Msh2-Msh6Molecular Cell, 2007
- How gene order is influenced by the biophysics of transcription regulationProceedings of the National Academy of Sciences of the United States of America, 2007
- Probing Transcription Factor Dynamics at the Single-Molecule Level in a Living CellScience, 2007
- Single-molecule analysis of 1D diffusion and transcription elongation of T7 RNA polymerase along individual stretched DNA moleculesNucleic Acids Research, 2007
- Single Molecule Measurements of Repressor Protein 1D Diffusion on DNAPhysical Review Letters, 2006
- DNA looping by two-site restriction endonucleases: heterogeneous probability distributions for loop size and unbinding forceNucleic Acids Research, 2006
- A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNAProceedings of the National Academy of Sciences, 2006
- Long-distance lateral diffusion of human Rad51 on double-stranded DNAProceedings of the National Academy of Sciences, 2006
- Strong physical constraints on sequence-specific target location by proteins on DNA moleculesNucleic Acids Research, 2006
- Kinetic Characterization of Linear Diffusion of the Restriction Endonuclease EcoRV on DNABiochemistry, 1998