Dissecting the role of the φ29 terminal protein DNA binding residues in viral DNA replication

Published by Oxford University Press on behalf of Nucleic Acids Research. Phage φ29 DNA replication takes place by a proteinpriming mechanism in which the viral DNA polymerase catalyses the covalent linkage of the initiating nucleotide to a specific serine residue of the terminal protein (TP). The N...

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Detalles Bibliográficos
Autores: Holguera, Isabel, Muñoz-Espín, D., Salas, Margarita
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2015
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/124306
Acceso en línea:http://hdl.handle.net/10261/124306
Access Level:acceso abierto
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spelling Dissecting the role of the φ29 terminal protein DNA binding residues in viral DNA replicationHolguera, IsabelMuñoz-Espín, D.Salas, MargaritaPublished by Oxford University Press on behalf of Nucleic Acids Research. Phage φ29 DNA replication takes place by a proteinpriming mechanism in which the viral DNA polymerase catalyses the covalent linkage of the initiating nucleotide to a specific serine residue of the terminal protein (TP). The N-terminal domain of the φ29 TP has been shown to bind to the host DNA in a sequence-independent manner and this binding is essential for the TP nucleoid localisation and for an efficient viral DNA replication in vivo. In the present work we have studied the involvement of the TP N-terminal domain residues responsible for DNA binding in the different stages of viral DNA replication by assaying the in vitro activity of purified TP N-terminal mutant proteins. The results show that mutation of TP residues involved in DNA binding affects the catalytic activity of the DNA polymerase in initiation, as the K<inf>m</inf> for the initiating nucleotide is increased when these mutant proteins are used as primers. Importantly, this initiation defect was relieved by using the φ29 double-stranded DNA binding protein p6 in the reaction, which decreased the K<inf>m</inf> of the DNA polymerase for dATP about 130-190 fold. Furthermore, the TP N-terminal domain was shown to be required both for a proper interaction with the DNA polymerase and for an efficient viral DNA amplification.Spanish Ministry of Economy and Competitiveness [BFU2011–23645] ; Consolider-Ingenio from the Spanish 2800 Nucleic Acids Research, 2015, Vol. 43, No. 5 Ministry of Science and Innovation [CSD2007–00015 to M.S.] ; Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular‘Severo Ochoa’. I.H. was holder of an FPU fellowship from the Spanish Ministry of Education. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2011- 23645]Peer ReviewedOxford University PressMinisterio de Ciencia e Innovación (España)Fundación Ramón ArecesMinisterio de Educación (España)Ministerio de Economía y Competitividad (España)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]20152015info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/124306reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)InglésSíinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1243062026-05-22T06:33:51Z
dc.title.none.fl_str_mv Dissecting the role of the φ29 terminal protein DNA binding residues in viral DNA replication
title Dissecting the role of the φ29 terminal protein DNA binding residues in viral DNA replication
spellingShingle Dissecting the role of the φ29 terminal protein DNA binding residues in viral DNA replication
Holguera, Isabel
title_short Dissecting the role of the φ29 terminal protein DNA binding residues in viral DNA replication
title_full Dissecting the role of the φ29 terminal protein DNA binding residues in viral DNA replication
title_fullStr Dissecting the role of the φ29 terminal protein DNA binding residues in viral DNA replication
title_full_unstemmed Dissecting the role of the φ29 terminal protein DNA binding residues in viral DNA replication
title_sort Dissecting the role of the φ29 terminal protein DNA binding residues in viral DNA replication
dc.creator.none.fl_str_mv Holguera, Isabel
Muñoz-Espín, D.
Salas, Margarita
author Holguera, Isabel
author_facet Holguera, Isabel
Muñoz-Espín, D.
Salas, Margarita
author_role author
author2 Muñoz-Espín, D.
Salas, Margarita
author2_role author
author
dc.contributor.none.fl_str_mv Ministerio de Ciencia e Innovación (España)
Fundación Ramón Areces
Ministerio de Educación (España)
Ministerio de Economía y Competitividad (España)
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
description Published by Oxford University Press on behalf of Nucleic Acids Research. Phage φ29 DNA replication takes place by a proteinpriming mechanism in which the viral DNA polymerase catalyses the covalent linkage of the initiating nucleotide to a specific serine residue of the terminal protein (TP). The N-terminal domain of the φ29 TP has been shown to bind to the host DNA in a sequence-independent manner and this binding is essential for the TP nucleoid localisation and for an efficient viral DNA replication in vivo. In the present work we have studied the involvement of the TP N-terminal domain residues responsible for DNA binding in the different stages of viral DNA replication by assaying the in vitro activity of purified TP N-terminal mutant proteins. The results show that mutation of TP residues involved in DNA binding affects the catalytic activity of the DNA polymerase in initiation, as the K<inf>m</inf> for the initiating nucleotide is increased when these mutant proteins are used as primers. Importantly, this initiation defect was relieved by using the φ29 double-stranded DNA binding protein p6 in the reaction, which decreased the K<inf>m</inf> of the DNA polymerase for dATP about 130-190 fold. Furthermore, the TP N-terminal domain was shown to be required both for a proper interaction with the DNA polymerase and for an efficient viral DNA amplification.
publishDate 2015
dc.date.none.fl_str_mv 2015
2015
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/124306
url http://hdl.handle.net/10261/124306
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Oxford University Press
publisher.none.fl_str_mv Oxford University Press
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,812429

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