Divalent metal dependence of site-specific DNA binding by EcoRV endonuclease

Measurements of binding equilibria of EcoRV endonuclease to DNA, for a series of base-analogue substrates, demonstrate that expression of sequence selectivity is strongly enhanced by the presence of Ca²⁺ ions. Binding constants were determined for short duplex oligodeoxynucleotides containing the cognate DNA site, three cleavable noncognate sites, and a fully nonspecific site. At pH 7.5 and 100 mM NaCl, the full range of specificity from the specific (tightest binding) to nonspecific (weakest binding) sites is 0.9 kcal/mol in the absence of metal ions and 5.8 kcal/mol in the presence of Ca²⁺. Precise determination of binding affinities in the presence of the active Mg²⁺ cofactor was found to be possible for substrates retaining up to 1.6% of wild-type activity, as determined by the rate of phosphoryl transfer. These measurements show that Ca²⁺ is a near-perfect analogue for Mg²⁺ in binding reactions of the wild-type enzyme with DNA base-analogue substrates, as it provides identical ΔΔG°(bind) values among the cleavable noncognate sites. Equilibrium dissociation constants of wild-type and base- analogue sites were also measured for the weakly active EcoRV mutant K38A, in the presence of either Mg²⁺ or Ca²⁺. In this case, Ca²⁺ allows expression of a greater degree of specificity than does Mg²⁺. ΔΔG°(bind) values of K38A toward specific versus nonspecific sites are 6.1 kcal/mol with Ca²⁺ and 3.9 kcal/mol with Mg²⁺, perhaps reflecting metal-specific conformational changes in the ground-state ternary complexes. The enhancement of binding specificity provided by divalent metal ions is likely to be general to many restriction endonucleases and other metal-dependent nucleic acid-modifying enzymes. These results strongly suggest that measurements of DNA binding affinities for EcoRV, and likely for many other restriction endonucleases, should be performed in the presence of divalent metal ions.