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

Abstract Restriction endonucleases of the PD…D/EXK family need Mg 2+ for DNA cleavage. Whereas Mg 2+ (or Mn 2+ ) promotes catalysis, Ca 2+ (without Mg 2+ ) only supports DNA binding. The role of Mg 2+ in DNA cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg 2+ involved in catalysis. To address this problem, we measured the Mg 2+ and Mn 2+ 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) Me 2+ per active site. DNA cleavage experiments were carried out at various Mg 2+ and Mn 2+ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg 2+ and Mn 2+ concentration dependence. In general, the Mg 2+ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn 2+ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg 2+ or Mn 2+ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca 2+ . Based on these results, we propose that one Mg 2+ or Mn 2+ is critical for restriction enzyme activation, and binding of a second Me 2+ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg 2+ or Mn 2+ mainly leads to an increase in K m , such that the inhibitory effect of excess Mg 2+ or Mn 2+ 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 Me 2+ binding to these enzymes.