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, Mónika; Pingoud, Alfred

doi:10.1016/j.jmb.2009.08.011

Cited by 62 publications

(96 citation statements)

References 94 publications

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“…As we could not reproduce this high-resolution DNA-complex crystal, we were not able to confirm this using the anomalous signal of Mn 2+ with longer wavelength X-rays, but the temperature factors of the refined metal sites suggested that the sites probably contained Mn 2+ and not Mg 2+ ( 2+ in EcoRV has been discussed based on the intrinsic affinities of these ions for phosphates and seems to be consistent with the observation that the Mn 2+ concentration optimum is about ten times lower than that of Mg 2+ for many PD-(D/E)xK-family endonucleases (Pingoud et al, 2009).…”

Section: +supporting

confidence: 60%

Structures of restriction endonucleaseHindIII in complex with its cognate DNA and divalent cations

Watanabe

Takasaki

Sato

et al. 2009

Acta Crystallogr D Biol Cryst

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Author(s) of this paper may load this reprint on their own web site or institutional repository provided that this cover page is retained. Republication of this article or its storage in electronic databases other than as specified above is not permitted without prior permission in writing from the IUCr.For further information see http://journals.iucr.org/services/authorrights.html Acta Crystallographica Section D: Biological Crystallography welcomes the submission of papers covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure-function papers that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such papers should present new insights into biology, chemistry or structure. Papers on crystallographic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis.Crystallography Journals Online is available from journals.iucr.org Acta Cryst. (2009 The three-dimensional crystal structures of HindIII bound to its cognate DNA with and without divalent cations were solved at 2.17 and 2.00 Å resolution, respectively. HindIII forms a dimer. The structures showed that HindIII belongs to the EcoRI-like (-class) subfamily of type II restriction endonucleases. The cognate DNA-complex structures revealed the specific DNA-recognition mechanism of HindIII by which it recognizes the palindromic sequence A/AGCTT. In the Mg 2+ ion-soaked structure the DNA was cleaved and two ions were bound at each active site, corresponding to the two-metal-ion mechanism.

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Section: +supporting

confidence: 60%

Structures of restriction endonucleaseHindIII in complex with its cognate DNA and divalent cations

Watanabe

Takasaki

Sato

et al. 2009

Acta Crystallogr D Biol Cryst

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“…This family of enzymes binds to one or two divalent metal ions, in particular, Mg 2ϩ or Mn 2ϩ . One of the ions is clearly involved in catalysis, whereas the role of the second one is not clearly established and may have a modulatory role (inhibition or stimulation, depending on the concentration and the nature of the ion) (17,26). PA-Nter shows endonuclease activity in the absence of the rest of the poly-merase and has the same metal ion dependence as the polymerase in intact RNPs.…”

mentioning

confidence: 99%

Mutational and Metal Binding Analysis of the Endonuclease Domain of the Influenza Virus Polymerase PA Subunit

Crépin

Dias

Palencia

et al. 2010

J Virol

136

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Influenza virus polymerase initiates the biosynthesis of its own mRNAs with capped 10-to 13-nucleotide fragments cleaved from cellular (pre-)mRNAs. Two activities are required for this cap-snatching activity: specific binding of the cap structure and an endonuclease activity. Recent work has shown that the cap-binding site is situated in the central part of the PB2 subunit and that the endonuclease activity is situated in the N-terminal domain of the PA subunit (PA-Nter). The influenza endonuclease is a member of the PD-(D/E)XK family of nucleases that use divalent metal ions for nucleic acid cleavage. Here we analyze the metal binding and endonuclease activities of eight PA-Nter single-point mutants. We show by calorimetry that the wild-type active site binds two Mn 2؉ ions and has a 500-fold higher affinity for manganese than for magnesium ions. The endonuclease activity of the isolated mutant domains are compared with the cap-dependent transcription activities of identical mutations in trimeric recombinant polymerases previously described by other groups. Mutations that inactivate the endonuclease activity in the isolated PA-Nter knock out the transcription but not replication activity in the recombinant polymerase. We confirm the importance of a number of active-site residues and identify some residues that may be involved in the positioning of the RNA substrate in the active site. Our results validate the use of the isolated endonuclease domain in a drug-design process for new anti-influenza virus compounds.

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“…The reaction is enhanced by the binding of metal ion at site B. A suppressive role of the second ion, as suggested by Pingoud et al (2009), is not in agreement with the observations made in the case of HindIII. This study reports the experimentally verified role of the two metal ions for the first time.…”

Section: Phosphodiester Bond Cleavagementioning

confidence: 55%

“…The number of metal ions retained at the active site in these structures varies; therefore, mechanisms with one metal ion, two metal ions and even three metal ions have independently been proposed (Pingoud et al, 2005). While kinetic analysis with computer simulations suggests that one metal ion is sufficient for the reaction to progress (Pingoud et al, 2009;Xie et al, 2008), it is still unclear whether the divalent metal ions in these multiple sites observed in the crystal structure are critical for the catalytic reaction of REases (Prasannan et al, 2010;Pingoud et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

“…For this, crystals of a HindIII-DNA complex were introduced into manganese-containing buffer and flash-cooled after soaking times of appropriate lengths. Some type IIP REases are fully activated by a tenfold lower concentration of manganese ion compared with magnesium ion (Pingoud et al, 2009). Furthermore, manganese ion is expected to be favoured at site A in HindIII (Watanabe et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

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Analysis of the HindIII-catalyzed reaction by time-resolved crystallography

Kawamura

Kobayashi

Watanabe

2015

Acta Cryst D Biol Crystallogr

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In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII-DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metalion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the twometal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

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

Cited by 62 publications

References 94 publications

Structures of restriction endonucleaseHindIII in complex with its cognate DNA and divalent cations

Structures of restriction endonucleaseHindIII in complex with its cognate DNA and divalent cations

Mutational and Metal Binding Analysis of the Endonuclease Domain of the Influenza Virus Polymerase PA Subunit

Analysis of the HindIII-catalyzed reaction by time-resolved crystallography

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