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- Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA

Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA

Darren M. Gowers,* Geoffrey G. Wilson, and Stephen E. Halford
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom
To whom correspondence should be addressed.
*Present address: IBBS Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, Portsmouth PO1 2DY, United Kingdom.
Present address: New England Biolabs, 240 County Road, Ipswich, MA 01938-2723.
Edited by Peter H. von Hippel, University of Oregon, Eugene, OR
Open Access Article : Proc Natl Acad Sci U S A. 2005 November 1; 102(44): 15883–15888.


Proteins that act at specific DNA sequences bind DNA randomly and then translocate to the target site. The translocation is often ascribed to the protein sliding along the DNA while maintaining continuous contact with it. Proteins also can move on DNA by multiple cycles of dissociation/reassociation within the same chain. To distinguish these pathways, a strategy was developed to analyze protein motion between DNA sites. The strategy reveals whether the protein maintains contact with the DNA as it transfers from one site to another by sliding or whether it loses contact by a dissociation/reassociation step. In reactions at low salt, the test protein stayed on the DNA as it traveled between sites, but only when the sites were <50 bp apart. Transfers of >30 bp at in vivo salt, and over distances of >50 bp at any salt, always included at least one dissociation step. Hence, for this enzyme, 1D sliding operates only over short distances at low salt, and 3D dissociation/reassociation is its main mode of translocation.
Keywords: DNA–protein interaction, recognition sequence, restriction enzyme
Genetic events such as replication, transcription, and restriction depend on proteins acting at specific DNA sites (15). The specific sequences constitute a minute fraction of genomic DNA so the protein is unlikely to collide with its target site by random diffusion through bulk solution (6, 7). Moreover, under in vivo conditions, proteins generally bind to specific sites at rates near the diffusion limit for protein–ligand associations, ≈1 × 108 M-1·s-1 (8). However, if a protein has to undergo many separate collisions with the DNA before hitting the target, the association rate could be reduced to far below this limit (9). [Protein–DNA association rates can seemingly exceed the diffusion limit (6, 10), but very rapid rates occur only at low salt and are mainly due to electrostatic factors (8).] Hence, the protein will first collide with a nonspecific site and then transfer to the specific site by an intramolecular process, by facilitated diffusion (6, 8, 9).

Three distinct pathways have been proposed for facilitated diffusion (811). In one, called “sliding,” the protein moves along the DNA while remaining in continuous contact with it, by a diffusional process confined to the linear contour of the DNA. The volume of space that the protein needs to search through is thus reduced to the 1D contour (9). However, the random directionality of thermal diffusion gives equal probabilities for forward and reverse steps, so the protein returns repeatedly to its start point (12). Consequently, the rate at which a protein locates its target site is accelerated if the protein slides over relatively short distances, up to ≈10 times the length of the target site, but 1D diffusion over longer distances slows down the rate of target site location (8).

In the second, “hopping/jumping,” the protein moves between sites through 3D space, by dissociating from its initial site before reassociating elsewhere in the same chain (8, 9, 11). After dissociating, the protein will spend some time in the vicinity of the initial site, because of its relatively small diffusion constant (6, 7), so most rebinding events will be short-range “hops” back to or near that site (11, 13). Such hops are distinct from 1D diffusion because even a 1-bp hop breaks the continuous contact between DNA and protein. Occasionally, the protein may “jump” to a new site many bp away from the starting site but that is still in the same chain rather than a different chain. The intramolecular nature of jumping is due to the distances between DNA molecules in solution being much larger than those between two segments of the same chain (8). Hence, a protein can diffuse away from the initial site yet still remain inside the domain of that DNA (14).

In a further scheme, “intersegmental transfer” (811), the protein moves from one site to another by transiently binding both at the same time; it then dissociates from one to leave itself on the other. Proteins that bind two sites do so most readily with sites ≈400 bp apart: shorter separations are impeded by the need to bend and/or twist the intervening DNA (15). Intersegmental transfer is thus limited to large steps on DNA by proteins that can bind two sites at the same time. It cannot apply to the system examined here, which involves translocations over short distances by a protein that binds one site at a time.

At present, facilitated diffusion is usually ascribed to 1D diffusion (16, 17). However, much of the experimental data that support sliding can be reconciled to other schemes. For example, the effects of DNA length on the kinetics and/or equilibria of the protein binding to its site are often correlated to 1D diffusion (1719). However, to distinguish the schemes, it is necessary to vary not only the length of the DNA but also the affinity of the protein for nonspecific DNA (8, 11), the latter normally being implemented by varying the salt concentration (20). Otherwise, the length dependency reveals that the nonspecific DNA is on the path to the specific site, but not the actual pathway (18). Observations of single protein molecules on DNA, by fluorescence or microscopy (2123), have likewise been reconciled to sliding, although the distinction between the schemes requires a time resolution of <1 ms [because the process may cover 103 bp/s (9)] and a spatial resolution of <1 nm (to see if the protein maintains contact as it takes 1-bp steps on the DNA). No current technique meets both requirements (24).

Conversely, several recent studies have shown that the transfer from nonspecific to specific sites involves multiple dissociation/reassociations (2527). For instance, the processivity of the EcoRV nuclease, its ability to cleave two recognition sites during one DNA-binding event, decreased as the separation of the sites was increased from 50 to 750 bp in a manner that excluded sliding from being the only route (25). Instead, the data matched best a hopping/jumping scheme but where each reassociation is followed by the protein scanning every sequence for ≈25 bp on either side of the new site. In further experiments on a 3.7-kb plasmid with one recognition site, EcoRV located the site more readily on the supercoiled plasmid than on its relaxed form, which shows that it depends on the 3D rather than the 1D distance to the site (26). The plasmid was converted into a catenane with two interlinked rings of DNA, one of 3.4 kb with only nonspecific sequences and one of 0.3 kb with the recognition site. The transfer from nonspecific to specific site occurred equally well on the catenane, where most of the nonspecific DNA is tethered to, but not contiguous with, the target, as on the plasmid (26). Hence, many proteins seem to translocate along DNA over distances of ≥50 bp primarily by hopping/jumping, but sliding may still play a role in short-range transfers over ≤50 bp. Several theoretical studies also have concluded that the fastest route to a target site in a long DNA chain involves a combination of hopping/jumping steps interspersed by 1D sliding over short distances (8, 12, 28, 29).

An unanswered question about target-site location by proteins on DNA is thus the nature of short-range translocations, over distances of <100 bp. We describe here a strategy that reveals whether the protein maintains continuous contact with the DNA as it translocates from one site to another or whether it loses track of the path of the DNA between the sites one or more times during the transfer: i.e., the fraction of translocation events completed solely by 1D diffusion and the fraction that involves at least one dissociation/reassociation step.


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