The Type III restriction enzyme EcoP15I is a heterotrimer of two Mod and one Res subunit. The Mod subunits recognize the sequence CAGCAG and may methylate the second adenine residue. The Res subunit is homologous to members of superfamily 2 helicases and translocases.
While modification of the DNA is possible with such a trimeric enzyme DNA cleavage needs the action of two trimers which had been bound to recognition sequences in head to head orientation on the DNA substrate. The distance between these sequences is not important because single molecule experiments suggest a mechanism in which one trimer after binding to the recognition sequence starts a one-dimensional diffusion along the DNA until it collides with a second trimer bound to the other recognition sequence. ATP hydrolysis by the Res subunit is necessary for conformational changes to the sliding conformation of the enzyme. The collision complex of two trimers then can cleave the DNA double strand by activating the endonuclease domains in the Res subunits.
Although a first structure of an EcorP15I trimer bound to DNA was solved many questions about the enzyme are not answered yet. In the crystal an AMP was bound to the Res domain. Therefore it is not clear whether it mimics the ATP or the ADP bound state of the enzyme. Furthermore, the endonuclease domain was not visible. Thus nothing can be said about the conformation necessary for cleavage. We have begun to address these questions by trying to solve the structures of additional complexes of the enzyme with several DNA substrates and nucleotide analogs.
This work is a cooperation with PD Dr. Monika Reuter (Charité, Berlin)