Visualizing phosphodiester-bond hydrolysis by an endonuclease

Rafael Molina; Stefano Stella; Pilar Redondo; Hansel Gomez; María José Marcaida; Modesto Orozco; Jesús Prieto; Guillermo Montoya

doi:10.1038/nsmb.2932

Visualizing phosphodiester-bond hydrolysis by an endonuclease

Rafael Molina, Stefano Stella, Pilar Redondo, Hansel Gomez, María José Marcaida, Modesto Orozco, Jesús Prieto, Guillermo Montoya

Protein Structure and Function Program

25 Citations (Scopus)

Abstract

The enzymatic hydrolysis of DNA phosphodiester bonds has been widely studied, but the chemical reaction has not yet been observed. Here we follow the generation of a DNA double-strand break (DSB) by the Desulfurococcus mobilis homing endonuclease I-DmoI, trapping sequential stages of a two-metal-ion cleavage mechanism. We captured intermediates of the different catalytic steps, and this allowed us to watch the reaction by 'freezing' multiple states. We observed the successive entry of two metals involved in the reaction and the arrival of a third cation in a central position of the active site. This third metal ion has a crucial role, triggering the consecutive hydrolysis of the targeted phosphodiester bonds in the DNA strands and leaving its position once the DSB is generated. The multiple structures show the orchestrated conformational changes in the protein residues, nucleotides and metals during catalysis.

Original language	English
Journal	Nature Structural and Molecular Biology
Volume	22
Issue number	1
Pages (from-to)	65-72
Number of pages	8
ISSN	1545-9993
DOIs	https://doi.org/10.1038/nsmb.2932
Publication status	Published - Jan 2015

Keywords

DNA
Desulfurococcaceae
Endonucleases
Esters
Hydrolysis
Models, Molecular
Phosphates
Protein Conformation

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10.1038/nsmb.2932

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title = "Visualizing phosphodiester-bond hydrolysis by an endonuclease",

abstract = "The enzymatic hydrolysis of DNA phosphodiester bonds has been widely studied, but the chemical reaction has not yet been observed. Here we follow the generation of a DNA double-strand break (DSB) by the Desulfurococcus mobilis homing endonuclease I-DmoI, trapping sequential stages of a two-metal-ion cleavage mechanism. We captured intermediates of the different catalytic steps, and this allowed us to watch the reaction by 'freezing' multiple states. We observed the successive entry of two metals involved in the reaction and the arrival of a third cation in a central position of the active site. This third metal ion has a crucial role, triggering the consecutive hydrolysis of the targeted phosphodiester bonds in the DNA strands and leaving its position once the DSB is generated. The multiple structures show the orchestrated conformational changes in the protein residues, nucleotides and metals during catalysis.",

keywords = "DNA, Desulfurococcaceae, Endonucleases, Esters, Hydrolysis, Models, Molecular, Phosphates, Protein Conformation",

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T1 - Visualizing phosphodiester-bond hydrolysis by an endonuclease

AU - Molina, Rafael

AU - Stella, Stefano

AU - Redondo, Pilar

AU - Gomez, Hansel

AU - Marcaida, María José

AU - Orozco, Modesto

AU - Prieto, Jesús

AU - Montoya, Guillermo

PY - 2015/1

Y1 - 2015/1

N2 - The enzymatic hydrolysis of DNA phosphodiester bonds has been widely studied, but the chemical reaction has not yet been observed. Here we follow the generation of a DNA double-strand break (DSB) by the Desulfurococcus mobilis homing endonuclease I-DmoI, trapping sequential stages of a two-metal-ion cleavage mechanism. We captured intermediates of the different catalytic steps, and this allowed us to watch the reaction by 'freezing' multiple states. We observed the successive entry of two metals involved in the reaction and the arrival of a third cation in a central position of the active site. This third metal ion has a crucial role, triggering the consecutive hydrolysis of the targeted phosphodiester bonds in the DNA strands and leaving its position once the DSB is generated. The multiple structures show the orchestrated conformational changes in the protein residues, nucleotides and metals during catalysis.

AB - The enzymatic hydrolysis of DNA phosphodiester bonds has been widely studied, but the chemical reaction has not yet been observed. Here we follow the generation of a DNA double-strand break (DSB) by the Desulfurococcus mobilis homing endonuclease I-DmoI, trapping sequential stages of a two-metal-ion cleavage mechanism. We captured intermediates of the different catalytic steps, and this allowed us to watch the reaction by 'freezing' multiple states. We observed the successive entry of two metals involved in the reaction and the arrival of a third cation in a central position of the active site. This third metal ion has a crucial role, triggering the consecutive hydrolysis of the targeted phosphodiester bonds in the DNA strands and leaving its position once the DSB is generated. The multiple structures show the orchestrated conformational changes in the protein residues, nucleotides and metals during catalysis.

KW - DNA

KW - Desulfurococcaceae

KW - Endonucleases

KW - Esters

KW - Hydrolysis

KW - Models, Molecular

KW - Phosphates

KW - Protein Conformation

U2 - 10.1038/nsmb.2932

DO - 10.1038/nsmb.2932

M3 - Journal article

C2 - 25486305

SN - 1545-9993

VL - 22

SP - 65

EP - 72

JO - Nature Structural and Molecular Biology

JF - Nature Structural and Molecular Biology

IS - 1

ER -

Visualizing phosphodiester-bond hydrolysis by an endonuclease

Abstract

Keywords

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