How DNA coiling enhances target localization by proteins

Abstract

Many genetic processes depend on proteins interacting with specific sequences on DNA. Despite the large excess of nonspecific DNA in the cell, proteins can locate their targets rapidly. After initial nonspecific binding, they are believed to find the target site by 1D diffusion ("sliding") interspersed by 3D dissociation/reassociation, a process usually referred to as facilitated diffusion. The 3D events combine short intrasegmental "hops" along the DNA contour, intersegmental "jumps" between nearby DNA segments, and longer volume "excursions." The impact of DNA conformation on the search pathway is, however, still unknown. Here, we show direct evidence that DNA coiling influences the specific association rate of EcoRV restriction enzymes. Using optical tweezers together with a fast buffer exchange system, we obtained association times of EcoRV on single DNA molecules as a function of DNA extension, separating intersegmental jumping from other search pathways. Depending on salt concentration, targeting rates almost double when the DNA conformation is changed from fully extended to a coiled configuration. Quantitative analysis by an extended facilitated diffusion model reveals that only a fraction of enzymes are ready to bind to DNA. Generalizing our results to the crowded environment of the cell we predict a major impact of intersegmental jumps on target localization speed on DNA.

Fig. 1.

Protein search pathways and experimental approach. (A) Facilitated diffusion model with sliding and hopping resulting in probing of already visited sites (oversampling) on straight DNA. (B) On coiled DNA the enzyme can be captured by a segment it has not yet visited, leading to an intersegmental jump and reduction of oversampling. Because of random polymer fluctuations a protein performing an intersegmental jump at instant 1 will, upon unbinding at instant 1′ have moved far away from the original segment, accelerating the search process. (C) Experimental approach. Single DNA molecules are held between two beads in optical tweezers. EcoRV cleavage rates are measured at different DNA extensions. The conformational freedom of the DNA at these extensions can be divided into three regimes: (entropically) stretched DNA (Top), globular DNA (relaxed coil) (Middle), and a DNA coil that is squeezed between the beads. (Bottom)

Fig. 2.

Typical data trace of a cleavage event. Displayed is the force on the DNA in the direction along the stretched molecule. Every second, the DNA is rapidly stretched to ≈5–10 pN, resulting in sharp positive force spikes (negative spikes are caused by the fast movement of the beads and do not represent a real force on the DNA). Disappearance of these spikes implies that the DNA molecule has been cleaved in the preceding second. The best estimate for this event is exactly halfway between the last spike and the next stretching attempt. We thus define the reaction time as the time between transportation of the DNA construct into the enzyme-containing flow channel (t = 0 in the graph) and last upward spike plus 0.5 s. The statistical error that is introduced in the measured cleavage time (≈0.5 s) averages out for the large number of data points.

Fig. 3.

Determination of DNA hydrolysis rate, obtained with 500 nM EcoRV in reaction buffer with 100 mM NaCl. Because in the optical tweezers experiments only the cleavage of the second strand is observed, the distribution of cleavage times shows a lag phase. The single exponential fit (dashed line) gives a strand cleavage rate k₁ of 0.60 ± 0.03 s⁻¹ for the second strand. Because of degeneracy, cleavage of the first strand should in principle be twice as fast. As a result, the rate of hydrolysis for both DNA strands is 0.40 ± 0.03 s⁻¹. The average time required for DNA cleavage is the inverse, 2.5 ± 0.2 s. The full distribution can also directly be fitted with such a two-step process (k₁[exp(−k₁t) − exp(−2k₁t)]). Doing so (solid line) yields a strand cleavage rate k₁ of 0.54 ± 0.03 s⁻¹, comparable to the rate found above.

Fig. 4.

EcoRV association rates to the single recognition site on linear DNA molecules as a function of DNA extension. In 100 mM NaCl the association rate is found to be maximal around a fractional extension a ≈0.22, and decreases rapidly for increasing extensions, reducing almost a factor of two as the DNA is stretched. This effect is attributed to loss of intersegmental jumping in the search process of EcoRV. At low fractional extensions the DNA coil is deformed by the beads, resulting in a lowered local density of DNA segments around the recognition site. At lower salt conditions (0 and 25 mM) this effect disappears. (Inset) Association rates in 150 mM NaCl are ≈10-fold lower for all DNA extensions. Each data point consists of a minimum of 30 measurements.

Fig. 5.

EcoRV association rates measured on coiled DNA as a function of salt concentration, showing an optimum at ≈60 mM NaCl. (At this particular concentration, DNA hydrolysis becomes the rate-limiting step.) For NaCl concentrations >100 mM, the association rate of EcoRV to the specific site decreases exponentially.

Fig. 6.

The effect of hydrolysis on the measured cleavage rates, performed on data taken in 100 mM NaCl. For increased statistics data from all DNA conformations are combined here. (A) The first part of the distribution of cleavage times (0.25-s time bins) shows a lag phase caused by the presence of DNA hydrolysis as a second step in the reaction. The dashed line is the single-exponential fit (to the full distribution), and the solid curve is the fit for a two-step process. The hydrolysis rate (for the cleavage of both DNA strands) obtained by the fit is 0.4 ± 0.1 s⁻¹, in accordance with the direct measurements of this parameter (Fig. 3). (B) Probability distribution of cleavage time, constructed from the same data as in A. Also in this representation a two-step process (solid line) describes the data more accurately than a single exponential (dashed line), signifying the presence of DNA hydrolysis (again with a rate of 0.4 ± 0.1 s⁻¹). (Inset) Probability distributions for data subsets with the smallest (black) and largest (gray) fractional extensions. Although this method may provide an “easy way out” for fitting both hydrolysis and association rate, the uncertainties of the fits are ill-defined, as the individual data points in this cumulative representation are not independent.