ϕ29 DNA polymerase active site: role of residue Val250 as Metal-dNTP complex ligand and in protein-primed initiation

DNA polymerases require two acidic residues to coordinate metal ions A and B at their polymerisation active site during catalysis of nucleotide incorporation. Crystallographic resolution of φ{symbol}29 DNA polymerase ternary complex showed that metal B coordination also depends on the carbonyl group...

Descripción completa

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:10486/709525
Acceso en línea:http://hdl.handle.net/10486/709525
https://dx.doi.org/10.1016/j.jmb.2009.10.061
Access Level:acceso abierto
Palabra clave:Metal Ligand
Polymerisation Active Site
Protein-Priming
Strand Displacement
ϕ29 DNA Polymerase
Biología y Biomedicina / Biología
id ES_b6c57d0f17de8bfcfc007f190e032c01
oai_identifier_str oai:repositorio.uam.es:10486/709525
network_acronym_str ES
network_name_str España
repository_id_str
spelling ϕ29 DNA polymerase active site: role of residue Val250 as Metal-dNTP complex ligand and in protein-primed initiationPérez Arnáiz, PatriciaLázaro, José M.Salas, Margaritade Vega, MiguelMetal LigandPolymerisation Active SiteProtein-PrimingStrand Displacementϕ29 DNA PolymeraseBiología y Biomedicina / BiologíaDNA polymerases require two acidic residues to coordinate metal ions A and B at their polymerisation active site during catalysis of nucleotide incorporation. Crystallographic resolution of φ{symbol}29 DNA polymerase ternary complex showed that metal B coordination also depends on the carbonyl group of Val250 that belongs to the highly conserved Dx2SLYP motif of eukaryotic-type (family B) DNA polymerases. In addition, multiple sequence alignments have shown the specific conservation of this residue among the DNA polymerases that use a protein as primer. Thus, to ascertain its role in polymerisation, we have analysed the behaviour of single mutations introduced at the corresponding Val250 of φ{symbol}29 DNA polymerase. The differences in nucleotide binding affinity shown by mutants V250A and V250F with respect to the wild-type DNA polymerase agree to a role for Val250 as a metal B-dNTP complex ligand. In addition, mutant V250F was severely affected in φ{symbol}29 DNA replication because of a large reduction in the catalytic efficiency of the protein-primed reactions. In the light of the φ{symbol}29 DNA polymerase structures, a role for Val250 residue in the maintenance of the proper architecture of the enzyme to perform the protein-primed reactions is also proposedThis work was supported by the Spanish Ministry of Science and Innovation grants BFU 2008-00215 to M.S. and PET2007-0160 to M.V., by the Autonomous Community of Madrid grant PMAT-0283-0505 to M.S., and by an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’ElsevierDepartamento de Biología MolecularFacultad de Ciencias20092009-10-31research articlehttp://purl.org/coar/resource_type/c_2df8fbb1AMhttp://purl.org/coar/version/c_ab4af688f83e57aainfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10486/709525https://dx.doi.org/10.1016/j.jmb.2009.10.061reponame: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:10486/7095252026-06-23T12:46:27Z
dc.title.none.fl_str_mv ϕ29 DNA polymerase active site: role of residue Val250 as Metal-dNTP complex ligand and in protein-primed initiation
title ϕ29 DNA polymerase active site: role of residue Val250 as Metal-dNTP complex ligand and in protein-primed initiation
spellingShingle ϕ29 DNA polymerase active site: role of residue Val250 as Metal-dNTP complex ligand and in protein-primed initiation
Pérez Arnáiz, Patricia
Metal Ligand
Polymerisation Active Site
Protein-Priming
Strand Displacement
ϕ29 DNA Polymerase
Biología y Biomedicina / Biología
title_short ϕ29 DNA polymerase active site: role of residue Val250 as Metal-dNTP complex ligand and in protein-primed initiation
title_full ϕ29 DNA polymerase active site: role of residue Val250 as Metal-dNTP complex ligand and in protein-primed initiation
title_fullStr ϕ29 DNA polymerase active site: role of residue Val250 as Metal-dNTP complex ligand and in protein-primed initiation
title_full_unstemmed ϕ29 DNA polymerase active site: role of residue Val250 as Metal-dNTP complex ligand and in protein-primed initiation
title_sort ϕ29 DNA polymerase active site: role of residue Val250 as Metal-dNTP complex ligand and in protein-primed initiation
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 Metal Ligand
Polymerisation Active Site
Protein-Priming
Strand Displacement
ϕ29 DNA Polymerase
Biología y Biomedicina / Biología
topic Metal Ligand
Polymerisation Active Site
Protein-Priming
Strand Displacement
ϕ29 DNA Polymerase
Biología y Biomedicina / Biología
description DNA polymerases require two acidic residues to coordinate metal ions A and B at their polymerisation active site during catalysis of nucleotide incorporation. Crystallographic resolution of φ{symbol}29 DNA polymerase ternary complex showed that metal B coordination also depends on the carbonyl group of Val250 that belongs to the highly conserved Dx2SLYP motif of eukaryotic-type (family B) DNA polymerases. In addition, multiple sequence alignments have shown the specific conservation of this residue among the DNA polymerases that use a protein as primer. Thus, to ascertain its role in polymerisation, we have analysed the behaviour of single mutations introduced at the corresponding Val250 of φ{symbol}29 DNA polymerase. The differences in nucleotide binding affinity shown by mutants V250A and V250F with respect to the wild-type DNA polymerase agree to a role for Val250 as a metal B-dNTP complex ligand. In addition, mutant V250F was severely affected in φ{symbol}29 DNA replication because of a large reduction in the catalytic efficiency of the protein-primed reactions. In the light of the φ{symbol}29 DNA polymerase structures, a role for Val250 residue in the maintenance of the proper architecture of the enzyme to perform the protein-primed reactions is also proposed
publishDate 2009
dc.date.none.fl_str_mv 2009
2009-10-31
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/10486/709525
https://dx.doi.org/10.1016/j.jmb.2009.10.061
url http://hdl.handle.net/10486/709525
https://dx.doi.org/10.1016/j.jmb.2009.10.061
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
_version_ 1869417474273837056
score 15,300719

Similares en LA Referencia