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

Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage, thereby introducing a loop in the DNA. We investi-gated looping dynamics of Type IIE restriction enzymes NaeI and NarI by tracking the Brownian motion of single tethered DNA molecules. DNA con-taining 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 loop-ing 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 compac-tion for higher enzyme concentrations. Addition of Ca21 increases the NaeI-DNA loop lifetime by two orders of magnitude and stimulates specific bind-ing by NarI. Finally, for both enzymes we observe exponentially distributed loop formation times, indi-cating that looping is dominated by (re)binding the second recognition site