The interplay between supercoiling and DNA modifying enzymes at the

DNA supercoiling refers to the overwinding or underwinding of the double helix, forming plait-like structures called plectonemes. Supercoiling is involved in all biological processes that occur on DNA. Despite this involvement, single-molecule in vitro studies on these processes largely work with relaxed DNA, overlooking the role supercoiling is playing in these processes. We have constructed 18-kb linear DNA substrates that can be tethered to a microscope coverslip at multiple sites, allowing the template to be topologically constrained. We can then use an intercalating dye to induce positive or negative supercoiling, which we can observe at the single-molecule level using TIRF microscopy. We directly visualise plectonemes diffusing along the DNA. However, in the presence of the restriction enzyme EcoRV, plectonemes remain stationary at the EcoRV binding site. We show that EcoRV has an increased cleavage efficiency in the presence of supercoiling, even though its binding efficiency is unaffected. These findings reveal a relationship between DNA topology and the activity of DNA modifying enzymes. We hypothesise that supercoiling makes cleavage more energetically favourable for restriction enzymes that bend DNA. This work reveals the kinetic interplay between supercoiling and enzyme activity, highlighting how DNA itself can modulate the processes that act upon it.