Short-range DNA translocation initiates long-range 1-dimensional DNA diffusion by the Type III restriction enzyme EcoP15I

Cleavage of bacteriophage DNA by the Type III Restriction-Modification enzymes requires long-range interaction between DNA sites. This is facilitated by 1-dimensional diffusion (“DNA sliding”) initiated by ATP hydrolysis catalysed by a superfamily 2 helicase-like ATPase. Here we combined ultrafast twist measurements based on plasmonic DNA origami nano-rotors with stopped-flow fluorescence and gel-based assays to examine the role(s) for ATP hydrolysis. Our data shows that the helicase-like domain plays multiple roles. Firstly, stabilising initial DNA-interactions alongside the methyltransferase subunits. Secondly, causing environmental changes in the flipped adenine base following hydrolysis of the first ATP. And thirdly, remodelling nucleoprotein interactions via constrained translocation of a ~5-20 bp dsDNA loop. Initiation of DNA sliding requires 10-15 bp DNA downstream of the motor, corresponding to the site of nuclease domain binding. Our data unifies previous contradictory communication models for Type III enzymes.