Translocation-independent dimerization of the EcoKI endonuclease visualized by atomic force microscopy

Bacterial type I restriction/modification systems are capable of performing multiple actions in response to the methylation pattern on their DNA recog nition sequences. The enzymes making up these systems serve to protect the bacterial cells against viral infection by binding to their recognition seq uences on the invading DNA and degrading it after extensive ATP-driven tran slocation, DNA cleavage has been thought to occur as the result of a collis ion between two translocating enzyme complexes. Using atomic force microsco py (AFM), we show here that EcoKI dimerizes rapidly when bound to a plasmid containing two recognition sites for the enzyme. Dimerization proceeds in the absence of ATP and is also seen with an EcoKI mutant (K477R) that is un able to translocate DNA. Only monomers are seen when the enzyme complex bin ds to a plasmid containing a single recognition site. Based on our results, we propose that the binding of EcoKI to specific DNA target sequences is a ccompanied by a conformational change that leads rapidly to dimerization. T his event is followed by ATP-dependent translocation and cleavage of the DN A.