Visual analysis of concerted cleavage by type IIF restriction enzyme SfiI in subsecond time region

Abstract

Many DNA regulatory factors require communication between distantly separated DNA sites for their activity. The type IIF restriction enzyme SfiI is often used as a model system of site communication. Here, we used fast-scanning atomic force microscopy to monitor the DNA cleavage process with SfiI and the changes in the single SfiI-DNA complex in the presence of either Mg²⁺ or Ca²⁺ at a scan rate of 1-2 fps. The increased time resolution allowed us to visualize the concerted cleavage of the protein at two cognate sites. The four termini generated by the cleavage were released in a multistep manner. The high temporal resolution enabled us to visualize the translocation of a DNA strand on a looped complex and intersegmental transfer of the SfiI protein in which swapping of the site is performed without protein dissociation. On the basis of our results, we propose that the SfiI tetramer can remain bound to one of the sites even after cleavage, allowing the other site on the DNA molecule to fill the empty DNA-binding cleft by combining a one-dimensional diffusion-mediated sliding and a segment transfer mechanism.

Figure 1

DNA and protein movement on a mica surface. (A) The initial frame of Movie S1. SfiI tetramers were indicated by yellow arrowheads. AFM imaging was performed in the presence of Ca²⁺. (B) Movement of DNA strands on a mica. The contours of four individual DNA strands were traced in every 20 frames and depicted in different colors. (C and D) Trajectories of SfiI protein on a mica. The particle position was plotted in x-y coordinates with the initial position as (0, 0). Trajectories in C and D correspond to the movements of the particles indicated by open arrowhead and solid arrow in A, respectively. (E) Mean-square displacement 〈Δr²〉 as a function of the interval time Δt. Mean-square displacement for trajectories (C) and (D) were shown by brown and blue squares, respectively. The straight lines represent linear fits to each trace.

Figure 2

Visualization of SfiI-DNA complex. (A) The locations of the two SfiI recognition sites along a 905 bp fragment are shown. (B) AFM image of SfiI-DNA complex obtained under a Ca²⁺-containing buffer. Loop and short arms are indicated by yellow and orange arrowheads, respectively. Scale bar: 100 nm. (C) Histograms of the contour length of full complex, loop, and arms (N = 34). Gaussian-fitted curve is overlaid on each histogram.

Figure 3

DNA cleavage by SfiI. (A) Successive time-lapse images of a SfiI-DNA reaction obtained at 2.0 fps in the presence of Mg²⁺. The elapsed time is indicated in each image. The image is cropped from the original scan size of 600 nm × 450 nm. Scale bar: 50 nm. (B) Schematic illustration of the DNA cleavage event.

Figure 4

Looping and translocation by SfiI. Successive time-lapse images of a SfiI-DNA complex obtained at 1.0 fps in the presence of Ca²⁺. The elapsed time is shown in each image. The image is cropped from the original scan size of 600 nm × 450 nm. (A) Diffusive looping and following translocation, before dissociation of the complex at 18 s. Scale bar: 50 nm. (B) Changes in DNA length over a time period of 13 s (from 0 to 13 s) measured in 1 s intervals. The contour lengths of the entire molecule, loops, arm 1, and arm 2 are indicated by, (○), (Δ), (×), and (⋄), respectively.

Figure 5

Segment transfer and formation of specific complex in trans. The experiment, same as Fig. 4, was performed with a different scanning rate. SfiI segment transfer event resulting in specific binding. Images were obtained at 2.0 fps. Scale bar: 50 nm.