Type III Restriction Enzymes Use 1D Diffusion to Communicate the Relative Orientation of their Distant Target Sites

Type III restriction enzymes sense the relative orientation of their distant target sites and cleave DNA only if at least two of them are situated in an inverted repeat. The communication process is strictly dependent on ATP hydrolysis catalyzed by their superfamily 2 helicase domains. Given the similarity to Type I restriction enzymes, which couple ATP hydrolysis to directed motion on DNA, unidirectional loop translocation that may partially be accompanied by 3D diffusive looping has been the suggested communication mechanism for Type III enzymes.Based on magnetic tweezers single-molecule cleavage experiments and ATPase measurements we suggest an alternative inter-site communication mechanism using 1D diffusion along the DNA contour (1). In order to verify this hypothesis we directly visualize the motion of quantum-dot labeled Type III restriction enzymes along DNA. For this we use a setup that combines magnetic tweezers with total internal reflection fluorescence microscopy. The enzymes undergo a fast diffusive motion along DNA capable of scanning kbp distances per second. We also find that the affinity of the enzymes to non-specific and specific DNA is regulated by the presence of ATP suggesting that ATP hydrolysis acts as a trigger for diffusion.Thus Type III restriction enzymes are the first DNA-modifying enzymes which communicate target site orientations over long distances via 1D diffusion.(1) van Aelst K, Toth J, Ramanathan SP, Schwarz FW, Seidel R, Szczelkun MD. Proc Natl Acad Sci U S A. 2010 May 18;107(20):9123-8.