Single-Molecule Studies Of ATP-Dependent Restriction Enzymes

Restriction enzymes (REs) are the central part of the defence system of bacteria against invading viruses. The protein complexes recognize viral DNA by the methylation state of their target sequence and destroy it by cleaving it into pieces. For this, the majority of REs need to interact with two distant target sites. This long-range inter-site communication can be accomplished either by passive 3D diffusive looping or by 1D motion along the DNA contour. Among the different classes of REs, Type I and Type III REs play a special role due to their helicase domains, which are key to the inter-site communication.For Type I REs it is well established that the helicase domain acts as a dsDNA translocating motor. Cleavage is triggered after a pure 1D communication process, when two translocating motors from distant target sites collide.In comparison, the communication mechanism for Type III REs has not been accurately defined and conflicting models including 3D diffusion and 1D translocation have been proposed. Using single-molecule DNA stretching based on magnetic tweezers, we provide evidence for a pure 1D communication mechanism in the absence of any 3D diffusive looping. Furthermore, we exclude translocation for inter-site communication due to the low ATPase rates and the observation that the enzymes move bidirectionally along DNA. From this we conclude that Type III REs use 1D diffusion to communicate between their distant target sites.In order to test the diffusion hypothesis we have started to track the movement of Type I and III REs along DNA using a setup combining magnetic tweezers with single-molecule fluorescence (total-internal reflection fluorescence microscopy).