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. 2013 May 7;104(9):2042-50.
doi: 10.1016/j.bpj.2013.03.030.

Obstacles may facilitate and direct DNA search by proteins

Amir Marcovitz  1 , Yaakov Levy
Affiliations

Obstacles may facilitate and direct DNA search by proteins

Amir Marcovitz et al. Biophys J. .

Abstract

DNA recognition by DNA-binding proteins (DBPs), which is a pivotal event in most gene regulatory processes, is often preceded by an extensive search for the correct site. A facilitated diffusion process in which a DBP combines three-dimensional diffusion in solution with one-dimensional sliding along DNA has been suggested to explain how proteins can locate their target sites on DNA much faster than predicted by three-dimensional diffusion alone. Although experimental and theoretical studies have recently advanced understanding of the biophysical principles underlying the search mechanism, the process under in vivo cellular conditions is poorly understood. In this study, we used various computational approaches to explore how the presence of obstacle proteins on the DNA influences search efficiency. At a low obstacle occupancy (i.e., when few obstacles occupy sites on the DNA), sliding by the searching DBP may be confined, which may impair search efficiency. The obstacles, however, can be bypassed during hopping events, and the number of bypasses is larger for higher obstacle occupancies. Dynamism on the part of the obstacles may even further facilitate search kinetics. Our study shows that the nature and efficiency of the search process may be governed not only by the intrinsic properties of the DBP and the salt concentration of the medium, but also by the in vivo association of DNA with other macromolecular obstacles, their location, and occupancy.

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Figures

Figure 1
Figure 1
Protein search mechanism on obstacle-covered DNA. (A) A schematic view of protein search mechanisms on DNA in the presence of obstacles (red rectangles). The protein may encounter an obstacle during sliding or may perform microscopic hops, which may assist bypassing the obstacles. DNA loops may also assist obstacle bypassing as the protein may occasionally use intersegmental transfers to traverse between two DNA regions that are far apart in sequence but are spatially close. (B) PP as a function of obstacle occupancy S (calculated as R/l, where R is the diameter of the searching protein and l is the length of spacer DNA between two adjacent obstacles, see inset). Obstacles repel the searching protein via electrostatic repulsions (DNA phosphate bead charge is modified from −1 to +1 at the location of the obstacles—shown by solid circles) or via excluded volume interactions (bulky obstacles with an excluded volume four times that of the protein, CEx/CEx0 = 4—shown by empty squares). Reported results reflect averages over 40–50 simulations under each salt condition and obstacle settings. Error bars reflect standard errors.
Figure 2
Figure 2
Protein confinement and obstacle bypassing. (A) Each 1D event (sliding and/or hopping dynamics), is characterized by two measures: dz, the distance between the minimal and maximal positions of the protein along the Z-axis and MSDz, the total mean square displacement of the protein along the Z-axis (diagrammed in the inset). Redundancy in 1D searches of DNA (MSDz/dz) is shown as a function of obstacle occupancy S for various salt concentrations for charged obstacles (solid circles) and for bulky obstacles (squares and diamonds) characterized by different ratios of the excluded volume repulsion radius (Cex) to the radius of DNA beads in nonoccupied sites (Cex0). (B) Productive hopping events versus S for the same salt concentrations and obstacle types as in (A). The inset shows the average duration of all hopping events 〈τhopping〉 (in units of simulation time steps) against the average number of hopping events per simulation for 0, 4, and 8 obstacles (corresponding to S = 0.04 (circles), 0.2 (triangles), or 0.4 (squares), respectively) for transient (unproductive) hops (upper diagram) and successful bypasses (lower diagram). (C) Protein sampling of DNA with a nonuniform obstacle occupancy including tightly packed inaccessible regions (S > ∼0.8), low occupancy oversampled regions (0 < S < ∼0.25), and moderately occupied efficiently sampled regions (∼0.25 < S < ∼0.8).
Figure 3
Figure 3
A typical trajectory of Sap-1 search on DNA associated with obstacles, under salt concentration of 0.03 M. For illustrative purposes, the obstacles (4 equally spaced charged obstacles) are shown as bulky spheres. Red dots mark the Cα position of residue 61 (from the center of the protein DNA recognition helix that is shown in green) during the simulation. The left and right conformations depict simulation snapshots where the protein propagates through helical sliding in which its recognition helix is situated at the DNA major groove. The two protein conformations in the middle depict bypass snapshots. Obstacle bypassing is enabled as the protein undergoes hopping and its recognition helix is excluded from the DNA.
Figure 4
Figure 4
Obstacle bypass kinetics. (A) Raw trajectories data of the displacement of the protein along the DNA axis (Z-axis) under salt concentrations of 0.01 M (blue) and 0.03 M (red). Upper panel: No obstacles (S = 0), middle panel: 4 obstacles (S = 0.2), low panel: 6 obstacles (S = 0.29). Dashed black lines denote the positions of obstacles. (B) Dependence of obstacle bypass kinetics (kbypass) on obstacle occupancy S at various salt concentrations for charged (circles) and bulky obstacles with Cex/Cex0 equals 2 (diamonds) or 4 (squares).
Figure 5
Figure 5
Protein-DNA energy landscape. (A) Protein-DNA energy landscape at two salt concentrations and under two obstacle occupancy conditions on DNA loaded with charged (smooth line) or bulky (dotted line) obstacles. (B) Monte Carlo simulation landscapes for a random walker on a 1D lattice showing a reduced energy barrier for bypassing under increasing obstacle occupancy.
Figure 6
Figure 6
Effect of mobile obstacles on DNA sampling efficiency. Lattice simulations of a protein (random walker) moving at a rate kProt,1D on a 1D lattice containing mobile obstacles. The protein may dissociate to the bulk at rate k1D-3D or reassociate at rate k3D-1D. The obstacles dissociate from their sites at a rate kObst. Immobile obstacles have a small μ value, dynamic obstacles have a large μ value, and ρ indicates the tendency of the protein to dissociate from the DNA and so corresponds to the salt concentration. (A and B) Lattice sampling efficiency (average of 10 simulations, normalized by the mean at 0% occupancy) under low salt (A, ρ = 10−10) and higher salt (B, ρ = 10−4) conditions. (C) As in C, but here the protein may hop over single obstacles with a probability of 0.5. The black lines in B and C, correspond to a sampling of obstacle-covered DNA with sampling efficiency similar to that achieved on naked DNA when obstacle bypassing are excluded (solid) or included (dashed).
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