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

Vera Pingoud, Wolfgang Wende, Peter Friedhoff, Monika Reuter, Jürgen Alves, Albert Jeltsch, Letif Mones, Monika Fuxreiter, Alfred Pingoud

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43 Citations (Scopus)

Abstract

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 Km, 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.

Original languageEnglish
Pages (from-to)140-160
Number of pages21
JournalJournal of molecular biology
Volume393
Issue number1
DOIs
Publication statusPublished - Oct 16 2009

Keywords

  • Mg
  • mechanism
  • nuclease
  • phosphodiester bond hydrolysis
  • restriction enzyme

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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    Pingoud, V., Wende, W., Friedhoff, P., Reuter, M., Alves, J., Jeltsch, A., Mones, L., Fuxreiter, M., & Pingoud, A. (2009). On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family. Journal of molecular biology, 393(1), 140-160. https://doi.org/10.1016/j.jmb.2009.08.011