Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI

Abstract

Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage, thereby introducing a loop in the DNA. We investigated looping dynamics of Type IIE restriction enzymes NaeI and NarI by tracking the Brownian motion of single tethered DNA molecules. DNA containing two endonuclease recognition sites spaced a few 100 bp apart connect small polystyrene beads to a glass surface. The position of a bead is tracked through video microscopy. Protein-mediated looping and unlooping is then observed as a sudden specific change in Brownian motion of the bead. With this method we are able to directly follow DNA looping kinetics of single protein-DNA complexes to obtain loop stability and loop formation times. We show that, in the absence of divalent cations, NaeI induces DNA loops of specific size. In contrast, under these conditions NarI mainly creates non-specific loops, resulting in effective DNA compaction for higher enzyme concentrations. Addition of Ca2+ increases the NaeI-DNA loop lifetime by two orders of magnitude and stimulates specific binding by NarI. Finally, for both enzymes we observe exponentially distributed loop formation times, indicating that looping is dominated by (re)binding the second recognition site.

Figure 1

Dimeric structure of NaeI in complex with two cognate DNA sites [(48), PDB ID: 1IAW]. The protein comprises two structurally different DNA-binding domains. Binding of cognate activator DNA to the ‘Topo’ domain functions as allosteric effector for binding and DNA cleavage by the ‘Endo’ domain (28,48). The two DNAs, shown in red, are bound under a 90° angle.

Figure 2

(a) DNA templates used in the experiments. Template #1 is 960 bp in length and has two NaeI recognition sites (and one NarI site). Template #2 is slightly longer, 1296 bp and harbors two NarI sites. In the looped state template #1 is 505 bp and template #2 991 bp in length. (b) Schematic representation of the experiment. Small beads are tethered with the DNA molecule in question to the glass slide. By tracking the x- and y-positions of the bead the magnitude of the Brownian motion is monitored, which is a measure for the tether length. Upon DNA loop formation by a restriction enzyme the Brownian motion of the bead suddenly decreases. This allows following DNA looping kinetics in real-time.

Figure 3

DNA looping by NaeI in the absence of divalent cations. (a) Typical data trace showing specific looping by NaeI (2 U/ml). Two distinct levels in the root mean square (RMS) amplitude of Brownian motion <R> can be recognized. The actual magnitudes of Brownian diffusion for the two states are estimated for each trace by fitting the histogram of <R> (shown on the right) to a double Gaussian, which agrees very well with the observed distribution. (b) Histograms of measured looped and unlooped state durations of NaeI (2 U/ml). As a result of the Gaussian filtering of the data with σ = 1.0 s, DNA loops with a lifetime shorter than 1.4 s cannot be reliably detected and are not taken into account. Left: NaeI–DNA looped complex lifetime. The data fits a single exponential (normalized χ² = 1.0) with lifetime τ_off = 18 ± 2 s. Right: NaeI-DNA loop formation. These data are reasonably well fitted by a single exponential (normalized χ² = 2.2), yielding a time constant τ_formation of 11 ± 2 s.

Figure 4

DNA looping by NarI in the absence of divalent cations. (a) Black trace: looping of DNA template #2 (two recognition sites) by NarI (2 U/ml). Grey trace: signal without protein. The histogram on the right clarifies even more that, in contrast to NaeI (Figure 3), it is not possible to distinguish two discrete levels of Brownian motion. (b) Black trace: non-specific looping by NarI (2 U/ml) on DNA template #1 (one site). The signal is similar to the trace observed with DNA template #2. Grey trace: signal without protein. (c) Effect of higher NarI concentrations. Addition of 10 U/ml NarI to DNA substrate #2 tethers results in a drop of Brownian motion from 170 nm to ∼110 nm over several minutes, presumably due to non-specific looping of multiple enzymes. After this initial process the average RMS motion of the bead fluctuates around this last value and never recovers toward the initial value. Upon addition of a high NarI concentration (100 U/ml), the Brownian motion is quickly reduced to a very low level due to non-specific DNA looping of many enzymes. This happens regardless of whether the DNA substrate has one or two NarI sites.

Figure 5

DNA looping by NarI in the presence of Ca²⁺ ions. (a) Data trace showing specific DNA looping by NarI in the presence of 2 mM Ca²⁺. The histogram on the right shows two levels of Brownian motion corresponding to the expected values for specific DNA looping. The two Gaussians are less separated than for NaeI, reflecting the smaller length of the specific NarI-loop. At times along the trace non-specific loops are formed. The small third peak at 120 nm presumably represents binding to a non-cognate site on this DNA template. This extra state was observed for several tethers. From time to time the bead transiently sticks to the surface (the Brownian motion drops to almost zero). (b) Histograms of measured looped and unlooped state durations of NarI (2 U/ml). Data are filtered with σ = 2.0 s, giving rise to a loop detection limit of 2.7 s. Left: distribution of the measured lifetimes of specific NarI DNA loops in the presence of Ca²⁺. The exponential fit gives a mean lifetime of 6 ± 1 s. (normalized χ² = 1.3) Right: distribution of the measured NarI DNA unlooped state durations in the presence of Ca²⁺. Again, the data fits a single exponential (normalized χ² = 0.8), yielding a mean loop formation time τ_formation of 43 ± 8 s.