Involvement of residues of the φ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase

Bacteriophage φ29 genome consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5′ end (TP-DNA) that together with a specific sequence constitutes the replication origins. To initiate replication, the DNA polymerase forms a heterodimer with a free TP that rec...

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Authors: Prado, Alicia del, Villar, Laurentino, Salas, Margarita
Format: article
Publication Date:2012
Country:España
Institution:Consejo Superior de Investigaciones Científicas (CSIC)
Repository:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/113643
Online Access:http://hdl.handle.net/10261/113643
Access Level:Open access
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spelling Involvement of residues of the φ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerasePrado, Alicia delVillar, LaurentinoSalas, MargaritaBacteriophage φ29 genome consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5′ end (TP-DNA) that together with a specific sequence constitutes the replication origins. To initiate replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the origins and initiates replication using as primer the hydroxyl group of TP residue Ser232. The 3D structure of the DNA polymerase/TP heterodimer allowed the identification of TP residues that could be responsible for interaction with the DNA polymerase. Here, we examined the role of TP residues Arg158, Arg169, Glu191, Asp198, Tyr250, Glu252, Gln253 and Arg256 by in vitro analyses of mutant derivatives. The results showed that substitution of these residues had an effect on either the stability of the TP/DNA polymerase complex (R158A) or in the functional interaction of the TP at the polymerization active site (R169A, E191A, Y250A, E252A, Q253A and R256A), affecting the first steps of φ29 TP-DNA replication. These results allow us to propose a role for these residues in the maintenance of the equilibrium between TP-priming domain stabilization and its gradual exit from the polymerization active site of the DNA polymerase as new DNA is being synthesized. © 2012 The Author(s).Spanish Ministry of Science and Innovation (grants BFU 2008-00215, and Consolider-Ingenio CSD2007-00015).; Autonomous Community of Madrid (grant P-MAT-0283-0505); Fundación Ramón ArecesPeer ReviewedOxford University PressMinisterio de Ciencia e Innovación (España)Comunidad de MadridFundación Ramón Areces2015201520122015info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501http://hdl.handle.net/10261/113643reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglésinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1136432026-05-22T06:33:51Z
dc.title.none.fl_str_mv Involvement of residues of the φ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase
title Involvement of residues of the φ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase
spellingShingle Involvement of residues of the φ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase
Prado, Alicia del
title_short Involvement of residues of the φ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase
title_full Involvement of residues of the φ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase
title_fullStr Involvement of residues of the φ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase
title_full_unstemmed Involvement of residues of the φ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase
title_sort Involvement of residues of the φ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase
dc.creator.none.fl_str_mv Prado, Alicia del
Villar, Laurentino
Salas, Margarita
author Prado, Alicia del
author_facet Prado, Alicia del
Villar, Laurentino
Salas, Margarita
author_role author
author2 Villar, Laurentino
Salas, Margarita
author2_role author
author
dc.contributor.none.fl_str_mv Ministerio de Ciencia e Innovación (España)
Comunidad de Madrid
Fundación Ramón Areces
description Bacteriophage φ29 genome consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5′ end (TP-DNA) that together with a specific sequence constitutes the replication origins. To initiate replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the origins and initiates replication using as primer the hydroxyl group of TP residue Ser232. The 3D structure of the DNA polymerase/TP heterodimer allowed the identification of TP residues that could be responsible for interaction with the DNA polymerase. Here, we examined the role of TP residues Arg158, Arg169, Glu191, Asp198, Tyr250, Glu252, Gln253 and Arg256 by in vitro analyses of mutant derivatives. The results showed that substitution of these residues had an effect on either the stability of the TP/DNA polymerase complex (R158A) or in the functional interaction of the TP at the polymerization active site (R169A, E191A, Y250A, E252A, Q253A and R256A), affecting the first steps of φ29 TP-DNA replication. These results allow us to propose a role for these residues in the maintenance of the equilibrium between TP-priming domain stabilization and its gradual exit from the polymerization active site of the DNA polymerase as new DNA is being synthesized. © 2012 The Author(s).
publishDate 2012
dc.date.none.fl_str_mv 2012
2015
2015
2015
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/113643
url http://hdl.handle.net/10261/113643
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
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 15.812455

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