LINEAR DIFFUSION OF THE RESTRICTION-ENDONUCLEASE ECORV ON DNA IS ESSENTIAL FOR THE IN-VIVO FUNCTION OF THE ENZYME

Linear diffusion along DNA is a mechanism of enhancing the association rates of proteins to their specific recognition sites on DNA. It has been demonstrated for several proteins in vitro, but to date in no cas e irt vivo, Here we show that the restriction endonuclease EcoRV slide s along the DNA, scanning similar to 1000 bp in one binding event, Thi s process is critically dependent on contacts between amino acid resid ues of the protein and the backbone of the DNA, The disruption of sing le hydrogen bonds and, in particular, the alteration of electrostatic interactions between amino acid side chains of the protein and phospha te groups of the DNA interfere with or abolish effective sliding, The efficiency of linear diffusion is dependent on salt concentration, hav ing a maximum at 50 mM NaCl. These results suggest that a nonspecific and mobile binding mode capable of linear diffusion is dependent on a subtle balance of forces governing the interaction of the enzyme and t he DNA. A strong correlation between the ability of EcoRV mutants to s lide along the DNA in vitro and to protect Escherichia coil cells from phage infection demonstrates that linear diffusion occurs in vivo and is essential for effective phage restriction.