Divalent metal ions at the active sites of the EcoRV and EcoRI restriction endonucleases

Restriction enzymes cannot cleave DNA without a metal ion cofactor. The specificities of the EcoRV and EcoRI endonucleases for metals were studied by measuring DNA cleavage rates with several metal ions and with combinations of metal ions. Both EcoRV and EcoRI had optimal activities with Mg2+, were less active with several other ions including Mn2+, and had virtually no activity with Ca2+. But the activities of EcoRV and EcoRI with either Mg2+ or Mn2+ were perturbed by Ca2+. For EcoRI, both Mg2+- and Mn(2+)-dependent activities, at both cognate and noncognate sites, were all inhibited by Ca2+. The activity of EcoRV at its recognition site with Mg2+ was also inhibited by Ca2+. But the Mn(2+)-dependent reaction at the EcoRV recognition site was stimulated by Ca2+. EcoRV activities at noncognate sites with either Mg2+ or Mn2+ displayed a biphasic response to Ca2+: stimulation at low concentrations of Ca2+ and inhibition at high concentrations. These observations, together with the known structures of the proteins, indicate that EcoRI needs only one metal ion per active site and is inactive when Mg2+ is displaced by Ca2+, while EcoRV needs two and that the displacement of one by Ca2+ can enhance activity. We propose a mechanism for phosphodiester hydrolysis by EcoRV that involves two metal ions.