KINETIC CHARACTERIZATION OF LINEAR DIFFUSION OF THE RESTRICTION-ENDONUCLEASE ECORV ON DNA

We have examined the kinetic parameters of linear diffusion of EcoRV o n DNA. The data were analyzed by Monte Carlo simulations in which the efficiency of recognition of EcoRV sites during linear diffusion, the efficiency of Linear diffusion, and, the behavior of enzymes at the en ds of linear DNA is explicitly treated, The analysis of the dependence of Linear diffusion on the concentrations of NaCl and MgCl2 shows tha t linear diffusion is maximal at 50 mM NaCl under all concentrations o f MgCl2 tested and increases with increasing concentrations of Mg2+ up to 10 mM. the highest concentration used in the test. Under these con ditions, EcoRV scans 2 x 10(6) bp during one binding event with a velo city of about 1.7 x 10(6) bp s(-1). The enzyme tends to overlook cleav age sites at 1 mM but not at 10 mM. MgCl2. This result confirms the th ermodynamic finding that EcoRV does not bind very specifically to DNA in the absence of Mg2+. It demonstrates that there is a Mg2+-dependent continuous transition between a nonspecific and a specific binding mo de of EcoRV to DNA, By comparing cleavage rates of linear DNA whose en ds are free or blocked, we have shown that EcoRV has a very low probab ility to fall off at the ends of linear DNA. The enzyme rather is ''re flected'' and continues linear diffusion. EcoRV does not cleave oligon ucleotides containing two EcoRV sites processively. Consequently, diss ociation of the enzyme from the cleavage products is not preceded by a transfer to nonspecific DNA, and linear diffusion is not involved in product dissociation in EcoRV.