On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family

Restriction endonucleases of the PD…D/EXK family need Mg²⁺ for DNA cleavage. Whereas Mg²⁺ (or Mn²⁺) promotes catalysis, Ca²⁺ (without Mg²⁺) only supports DNA binding. The role of Mg²⁺ in DNA cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg²⁺ involved in catalysis. To address this problem, we measured the Mg²⁺ and Mn²⁺ concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me²⁺ per active site. DNA cleavage experiments were carried out at various Mg²⁺ and Mn²⁺ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg²⁺ and Mn²⁺ concentration dependence. In general, the Mg²⁺ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn²⁺ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg²⁺ or Mn²⁺ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca²⁺. Based on these results, we propose that one Mg²⁺ or Mn²⁺ is critical for restriction enzyme activation, and binding of a second Me²⁺ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg²⁺ or Mn²⁺ mainly leads to an increase in K_m, such that the inhibitory effect of excess Mg²⁺ or Mn²⁺ can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me²⁺ binding to these enzymes.