DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway - PubMed (original) (raw)

DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway

Bram van den Broek et al. Nucleic Acids Res. 2005.

Abstract

Type II restriction endonucleases protect bacteria against phage infections by cleaving recognition sites on foreign double-stranded DNA (dsDNA) with extraordinary specificity. This capability arises primarily from large conformational changes in enzyme and/or DNA upon target sequence recognition. In order to elucidate the connection between the mechanics and the chemistry of DNA recognition and cleavage, we used a single-molecule approach to measure rate changes in the reaction pathway of EcoRV and BamHI as a function of DNA tension. We show that the induced-fit rate of EcoRV is strongly reduced by such tension. In contrast, BamHI is found to be insensitive, providing evidence that both substrate binding and hydrolysis are not influenced by this force. Based on these results, we propose a mechanochemical model of induced-fit reactions on DNA, allowing determination of induced-fit rates and DNA bend angles. Finally, for both enzymes a strongly decreased association rate is obtained on stretched DNA, presumably due to the absence of intradomain dissociation/re-association between non-specific sites (jumping). The obtained results should apply to many other DNA-associated proteins.

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Figures

Figure 1

The reaction pathway for orthodox type IIP restriction endonucleases. The association rate _k_on is the only rate that depends on enzyme concentration. Under normal conditions, the induced-fit rate (_k_ind) is much faster than hydrolysis (_k_hydr) and product dissociation (_k_diss). The applied tension F opposes DNA bending by the enzyme in the induced-fit process.

Figure 2

Crystal structures of specific enzyme–DNA complexes of BamHI (1) (23) and EcoRV (2) (2,18). The DNA configuration within both complexes is shown separately, (3) and (4), respectively. While BamHI does not distort its recognition site, EcoRV induces a 50° kink located at the center base pair step.

Figure 3

Schematic representation of the experimental approach. DNA (orange) is stretched between two beads (blue) trapped in optical tweezers. Enzymes (green spheres) in the solution diffuse in search of the recognition sequence (red).

Figure 4

Typical data trace of a cleavage event. 1: Tension is being applied (in this case ∼50 pN) and measured by the displacement of one of the trapped beads. 2: Start of enzyme flow. The drag force displaces the bead further from the center of the trap. The tension on the DNA molecule does not change, since both beads are influenced in the same way by the flow. 3: The flow is turned off. 4: The DNA is cleaved. Both beads recoil to the centers of the optical traps, instantly reducing the measured force to zero.

Figure 5

Measured cleavage rate versus DNA tension. (a) The effect of tension on cleavage of linearized pCco5 by EcoRV (number of recognition sites n = 1). EcoRV concentration was 25 nM in terms of dimers. The total amount of cutting events was 68. (b) EcoRV on Lambda phage DNA (n = 21), 32 cutting events. (c) Tension dependence on DNA cleavage by BamHI (300 nM) on the pCco5 derivative containing a single recognition site (squares, 78 events) and on Lambda phage DNA with five BamHI sites (triangles, 28 events) using 2.5 nM BamHI. Each point consists of at least 6 cleavage events. Vertical error bars represent the standard error of the mean rate. Horizontal error bars are the standard deviation of the combined DNA tensions (a result of the binning). The data in (a and b) are fitted to the described model (normalized χ2 are 0.6 and 0.2, respectively). The BamHI rates in (c) do not significantly vary with DNA tension and were fitted with constant values [normalized χ2 are 0.8 (hydrolysis, green line) and 1.6 (diffusion, blue line)].

Figure 6

Mechanochemical model of tension dependence. (a) Schematic representation of DNA bending by a restriction enzyme under an applied external force. The enzyme induces a sharp kink in the DNA at the center of the recognition sequence. α0 is the half bend angle θ/2. (b) Theoretical model curves representing the effect of tension on the induced-fit rate using a bend angle θ = 50°. Dashed curve: overcoming the enthalpic stretching of the recognition sequence. Dotted curve: local shortening of the DNA end-to-end distance due to a kink at the center of the recognition site. Dashed–dotted curve: additional bending and end-to-end shortening of the DNA protruding from the enzyme–DNA complex. Solid curve: all three effects added, providing the dependence of the induced-fit rate on DNA tension.

Figure 7

EcoRV concentration dependence of the cleavage rate. (a) Cleavage of pCco5 DNA (one recognition sequence) by EcoRV for different enzyme concentrations. Cleavage rates were obtained at low DNA tensions (<10 pN) to ensure that the induced-fit mechanism was not rate-limiting. A total of 157 events were measured. Error bars represent the standard error of the mean rate for each of the concentrations. (b) Distribution of the single-molecule cleavage reaction times of the averaged data point indicated with asterisk in (a) (51 cleavage events). The EcoRV concentration (500 nM) is such that hydrolysis only is the rate-limiting step. Hence, an exponential distribution is expected. This is indeed observed. The hydrolysis rate obtained by fitting an exponential (solid curve) is 0.27 ± 0.05 s−1 (χ2 = 0.7). (c) Histogram of cleavage events of the averaged data point indicated with hash in (a) (73 reactions). This distribution displays a two-step process. At 25 nM EcoRV association and hydrolysis are of the same order of magnitude. Both processes by themselves obey Poissonian statistics and follow exponential distributions. However, as hydrolysis can only occur after binding, they are not independent. The distribution is thus fit with a convolution of two exponentials (χ2 = 0.7). The rates found for association and hydrolysis are 0.13 ± 0.03 s−1 [_k_on = (5 ± 1)·106 M−1 s−1] and 0.29 ± 0.09 s−1, respectively. Both rates are identical to the rates found by fitting Equation 5 to the data points in (a).

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DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway - PubMed (original) (raw)