On the divalent metal ion dependence of DNA cleavage by restriction endonucleases of the EcoRI family

J Mol Biol. 2009 Oct 16;393(1):140-60. doi: 10.1016/j.jmb.2009.08.011. Epub 2009 Aug 13.

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 approximately 1 and 10 mM) is higher than the Mn(2+) concentration optimum (between approximately 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cations / pharmacology*
  • Coenzymes / pharmacology*
  • DNA Cleavage*
  • DNA Restriction Enzymes / metabolism*
  • Kinetics
  • Magnesium / pharmacology*
  • Manganese / pharmacology*

Substances

  • Cations
  • Coenzymes
  • Manganese
  • DNA Restriction Enzymes
  • Magnesium