Untersuchung des DNA-Schneidemechanismus von Typ l Restriktionsenzymen

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Restriction enzymes are the main defence mechanism of bacteria against invading viruses. They recognize viral DNA upon the methylation state of their target sequence and cleaving it into pieces. Type I restriction enzymes consist of a methyltransferase core unit, responsible for DNA binding, which can bind up to two motor subunits. On viral DNA target sites, these motor subunits start to bind and translocate adjacent DNA in an ATP-driven manner, which leads to the formation of large DNA loops. Cleavage occurs randomly, distant from the target sites and is thought to occur upon collision with a second translocating enzyme. While the cleavage mechanism of Type II restriction enzymes is quite well understood, little is so far known on how Type I restriction enzymes cleave DNA. Within this project we therefore want to investigate the cleavage mechanism of these enzymes. Using state-of-the-art single-molecule techniques, in particular magnetic tweezers combined with fluorescence microscopy, we want to directly observe individual enzymes complexes during translocation and cleavage to directly test and explore the collision model.