Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases

Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg2+ is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca2+ (alone) only supports DNA binding. To investigate the role of Mg2+ in DNA cleavage by restriction endonucleases, we have studied the Mg2+ and Mn2+ concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg2+ and Mn2+ concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20 mM while no activity was detected in the presence of Mn2+ and in the presence of Ca2+ cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn2+ than in the presence of Mg2+ and can be activated by Ca2+. Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a Km value for pTrcHisB DNA of 6.15 nM and a Vmax of 1.79 × 10−2 nM min−1, while EhoI had a Km for pUC19 plasmid of 8.66 nM and a Vmax of 2 × 10−2 nM min−1.