Functional importance of bacteriophage ϕ29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3′-5′ exonuclease active site

Recent crystallographic resolution of φ29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx2h motif of proofreading family B DNA polymerases. Single sub...

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Detalles Bibliográficos
Autores: Pérez Arnáiz, Patricia, Lázaro, José M., Salas, Margarita, de Vega, Miguel
Tipo de recurso: artículo
Fecha de publicación:2009
País:España
Institución:Universidad Autónoma de Madrid
Repositorio:Biblos-e Archivo. Repositorio Institucional de la UAM
Idioma:inglés
OAI Identifier:oai:repositorio.uam.es:10486/709542
Acceso en línea:http://hdl.handle.net/10486/709542
https://dx.doi.org/10.1016/j.jmb.2009.06.068
Access Level:acceso abierto
Palabra clave:ϕ29 DNA Polymerase
3′-5′ Exonuclease
Site-Directed Mutagenesis
ssDNA Binding
Strand Displacement
Biología y Biomedicina / Biología
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spelling Functional importance of bacteriophage ϕ29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3′-5′ exonuclease active sitePérez Arnáiz, PatriciaLázaro, José M.Salas, Margaritade Vega, Miguelϕ29 DNA Polymerase3′-5′ ExonucleaseSite-Directed MutagenesisssDNA BindingStrand DisplacementBiología y Biomedicina / BiologíaRecent crystallographic resolution of φ29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx2h motif of proofreading family B DNA polymerases. Single substitution of φ29 DNA polymerase residue Tyr148 to Ala rendered an enzyme with a reduced capacity to stabilize the binding of the primer terminus at the 3′-5′ exonuclease active site, not having a direct role in the catalysis of the reaction. Analysis of the 3′-5′ exonuclease on primer/template structures showed a critical role for residue Tyr148 in the proofreading of DNA polymerisation errors. In addition, Tyr148 is not involved in coupling polymerisation to strand displacement in contrast to the catalytic residues responsible for the exonuclease reaction, its role being restricted to stabilisation of the frayed 3′ terminus at the exonuclease active site. Altogether, the results lead us to extend the consensus sequence of the above motif of proofreading family B DNA polymerases into Kx2hxA. The different solutions adopted by proofreading DNA polymerases to stack the 3′ terminus at the exonuclease site are discussed. In addition, the results obtained with mutants at φ29 DNA polymerase residue Pro129 allow us to rule out a functional role as ssDNA ligand for this residueThis work has been aided by the Spanish Ministry of Science and Innovation grant BFU 2008-00215, by the Autonomous Community of Madrid grant PMAT-0283-0505 and by an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’ElsevierDepartamento de Biología MolecularFacultad de Ciencias20092009-07-01research articlehttp://purl.org/coar/resource_type/c_2df8fbb1AMhttp://purl.org/coar/version/c_ab4af688f83e57aainfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10486/709542https://dx.doi.org/10.1016/j.jmb.2009.06.068reponame:Biblos-e Archivo. Repositorio Institucional de la UAMinstname:Universidad Autónoma de MadridInglésengopen accesshttp://purl.org/coar/access_right/c_abf2info:eu-repo/semantics/openAccessoai:repositorio.uam.es:10486/7095422026-06-23T12:46:27Z
dc.title.none.fl_str_mv Functional importance of bacteriophage ϕ29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3′-5′ exonuclease active site
title Functional importance of bacteriophage ϕ29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3′-5′ exonuclease active site
spellingShingle Functional importance of bacteriophage ϕ29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3′-5′ exonuclease active site
Pérez Arnáiz, Patricia
ϕ29 DNA Polymerase
3′-5′ Exonuclease
Site-Directed Mutagenesis
ssDNA Binding
Strand Displacement
Biología y Biomedicina / Biología
title_short Functional importance of bacteriophage ϕ29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3′-5′ exonuclease active site
title_full Functional importance of bacteriophage ϕ29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3′-5′ exonuclease active site
title_fullStr Functional importance of bacteriophage ϕ29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3′-5′ exonuclease active site
title_full_unstemmed Functional importance of bacteriophage ϕ29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3′-5′ exonuclease active site
title_sort Functional importance of bacteriophage ϕ29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3′-5′ exonuclease active site
dc.creator.none.fl_str_mv Pérez Arnáiz, Patricia
Lázaro, José M.
Salas, Margarita
de Vega, Miguel
author Pérez Arnáiz, Patricia
author_facet Pérez Arnáiz, Patricia
Lázaro, José M.
Salas, Margarita
de Vega, Miguel
author_role author
author2 Lázaro, José M.
Salas, Margarita
de Vega, Miguel
author2_role author
author
author
dc.contributor.none.fl_str_mv Departamento de Biología Molecular
Facultad de Ciencias
dc.subject.none.fl_str_mv ϕ29 DNA Polymerase
3′-5′ Exonuclease
Site-Directed Mutagenesis
ssDNA Binding
Strand Displacement
Biología y Biomedicina / Biología
topic ϕ29 DNA Polymerase
3′-5′ Exonuclease
Site-Directed Mutagenesis
ssDNA Binding
Strand Displacement
Biología y Biomedicina / Biología
description Recent crystallographic resolution of φ29 DNA polymerase complexes with ssDNA at its 3′-5′ exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx2h motif of proofreading family B DNA polymerases. Single substitution of φ29 DNA polymerase residue Tyr148 to Ala rendered an enzyme with a reduced capacity to stabilize the binding of the primer terminus at the 3′-5′ exonuclease active site, not having a direct role in the catalysis of the reaction. Analysis of the 3′-5′ exonuclease on primer/template structures showed a critical role for residue Tyr148 in the proofreading of DNA polymerisation errors. In addition, Tyr148 is not involved in coupling polymerisation to strand displacement in contrast to the catalytic residues responsible for the exonuclease reaction, its role being restricted to stabilisation of the frayed 3′ terminus at the exonuclease active site. Altogether, the results lead us to extend the consensus sequence of the above motif of proofreading family B DNA polymerases into Kx2hxA. The different solutions adopted by proofreading DNA polymerases to stack the 3′ terminus at the exonuclease site are discussed. In addition, the results obtained with mutants at φ29 DNA polymerase residue Pro129 allow us to rule out a functional role as ssDNA ligand for this residue
publishDate 2009
dc.date.none.fl_str_mv 2009
2009-07-01
dc.type.none.fl_str_mv research article
http://purl.org/coar/resource_type/c_2df8fbb1
AM
http://purl.org/coar/version/c_ab4af688f83e57aa
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10486/709542
https://dx.doi.org/10.1016/j.jmb.2009.06.068
url http://hdl.handle.net/10486/709542
https://dx.doi.org/10.1016/j.jmb.2009.06.068
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:Biblos-e Archivo. Repositorio Institucional de la UAM
instname:Universidad Autónoma de Madrid
instname_str Universidad Autónoma de Madrid
reponame_str Biblos-e Archivo. Repositorio Institucional de la UAM
collection Biblos-e Archivo. Repositorio Institucional de la UAM
repository.name.fl_str_mv
repository.mail.fl_str_mv
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