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, M. Fuxreiter, Alfred Pingoud

Research output: Contribution to journalArticle

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

Fingerprint

Deoxyribonuclease EcoRI
DNA Cleavage
DNA Restriction Enzymes
Metals
Ions
Catalysis
Catalytic Domain
Enzymes
Enzyme Activation
Molecular Dynamics Simulation
Osmolar Concentration
DNA

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Pingoud, V., Wende, W., Friedhoff, P., Reuter, M., Alves, J., Jeltsch, A., ... 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

On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family. / Pingoud, Vera; Wende, Wolfgang; Friedhoff, Peter; Reuter, Monika; Alves, Jürgen; Jeltsch, Albert; Mones, Letif; Fuxreiter, M.; Pingoud, Alfred.

In: Journal of Molecular Biology, Vol. 393, No. 1, 16.10.2009, p. 140-160.

Research output: Contribution to journalArticle

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, vol. 393, no. 1, pp. 140-160. https://doi.org/10.1016/j.jmb.2009.08.011
Pingoud, Vera ; Wende, Wolfgang ; Friedhoff, Peter ; Reuter, Monika ; Alves, Jürgen ; Jeltsch, Albert ; Mones, Letif ; Fuxreiter, M. ; Pingoud, Alfred. / On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family. In: Journal of Molecular Biology. 2009 ; Vol. 393, No. 1. pp. 140-160.
@article{c11ad911056546e592f5a8635f0ef4a8,
title = "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 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.",
keywords = "mechanism, Mg, nuclease, phosphodiester bond hydrolysis, restriction enzyme",
author = "Vera Pingoud and Wolfgang Wende and Peter Friedhoff and Monika Reuter and J{\"u}rgen Alves and Albert Jeltsch and Letif Mones and M. Fuxreiter and Alfred Pingoud",
year = "2009",
month = "10",
day = "16",
doi = "10.1016/j.jmb.2009.08.011",
language = "English",
volume = "393",
pages = "140--160",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family

AU - Pingoud, Vera

AU - Wende, Wolfgang

AU - Friedhoff, Peter

AU - Reuter, Monika

AU - Alves, Jürgen

AU - Jeltsch, Albert

AU - Mones, Letif

AU - Fuxreiter, M.

AU - Pingoud, Alfred

PY - 2009/10/16

Y1 - 2009/10/16

N2 - 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.

AB - 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.

KW - mechanism

KW - Mg

KW - nuclease

KW - phosphodiester bond hydrolysis

KW - restriction enzyme

UR - http://www.scopus.com/inward/record.url?scp=70349229163&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70349229163&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2009.08.011

DO - 10.1016/j.jmb.2009.08.011

M3 - Article

C2 - 19682999

AN - SCOPUS:70349229163

VL - 393

SP - 140

EP - 160

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 1

ER -