The effect of salt on the binding of the eucaryotic DNA nicking-closing enzyme to DNA and chromatin

Abstract

The optimum monovalent cation concentration (Na⁺ or K⁺) for the relaxation of superhelical DNA by the rat liver nicking-closing enzyme under conditions of DNA excess was found to be 150–200 mM. The detection of a nicked DNA species after stopping a reaction with alkali depends on having a high molar ratio of enzyme to DNA and is maximal between 50 and 100 mM monovalen cation. Varying the salt concentration from 15 to 200 mM appears to have no effect on the catalysis of the nicking-closing reaction by the enzyme. Instead different salt optima in these two assays can be explained by the observation that the nicking-closing enzyme acts by a processive mechanism below 100 mM salt and becomes nonprocessive above 150 mM. The salt elution of the nicking-closing enzyme from resting cell chromatin appears to be similar to that which one would expect for the elution of the enzyme from naked DNA. However, greater than 70% of the chromatin associated enzyme activity remained bound to chromatin from growing cells at 300 mM salt, a concentration at which there is no significant binding to naked DNA in vitro.