Sliding and jumping of single EcoRV restriction enzymes on non-cognate DNA

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.

Protein–DNA Electrostatics

“…These experiments confirm that proteins may slide along DNA and generally display a standard diffusion dynamics. Experiments also show that the sliding lifetime is sensitive to the salt concentration [1,11,13]. This supports the idea that electrostatics is involved to some extent in the intermittent behavior, with a probable role for the dissolved salt ions.…”

“…However, experimentalists clearly distinguish at least phases where the proteins are "on" DNA (and can therefore be observed) from phases where the protein dissociates from it. Moreover, rapid displacements along a same DNA molecule have been observed [13] that cannot be compatible with pure 1D diffusion along the double helix. The question therefore arise of how these different protein states or modes of displacement can be accounted for in the context of a continuous description.…”

“…Fluorescence microscopy is used to study processes on large spatial scales and temporal areas (from nanometer to micrometer and from nanosecond to second) [11,44,45,13,46,47]. The operating principle is simple: the protein is chemically linked to a fluorescent label (organic fluorophores, fluorescent nano-crystals, fluorescent proteins, quantum dots ...) and can therefore be observed optically.…”

“…An example of the results obtained by fluorescence microscopy is represented by the work of Pierre Desbiolles group [13], an extract of which is given in Figure 4. The registration of the position of the endonuclease EcoRV when bound non-specifically to DNA is decomposed into a longitudinal component and a transverse component.…”

“…The existence of 1D diffusion or sliding has then been proved by several experiments [7,8,9] and in particular by fluorescence microscopy [10,11,12,13]. In this kind of experiments the two extremities of a DNA molecule are bound on a surface, in such a manner that the DNA is softly stretched.…”