Facilitated diffusion of restriction enzyme EcoRV along DNA in crowded, confined conditions

Protein mobility is a central regulator of essential cellular processes. Here, we reveal how macromolecular crowding by dextran nanoparticles reshapes the facilitated diffusion of the restriction enzyme EcoRV by tracking its motion along single DNA molecules using fluorescence microscopy. DNA was confined in nanofluidic channels with a 60 nm cross-section, a regime in which it exhibits transverse undulations and wall deflections. Above a critical dextran concentration, DNA molecules collapse into a condensed, compact state. Prior to this collapse, crowding dramatically reduced EcoRV mobility by limiting dissociation from DNA and constraining long-range jumps due to increased medium viscosity. Together with regulatory factors such as cognate site recognition, DNA methylation, and ionic conditions, these findings provide mechanistic insights into DNA target search and reveal how nanoscale crowding shapes chromosome organization.