Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs

Bacteriophage φ29 DNA polymerase is a unique enzyme endowed with two distinctive properties, high processivity and faithful polymerization coupled to strand displacement, that have led to the development of protocols to achieve isothermal amplification of limiting amounts of both circular plasmids a...

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Detalles Bibliográficos
Autores: Vega, Miguel de, Lázaro, José M., Mencía, Mario, Blanco Dávila, Luis, Salas, Margarita
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2010
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/182852
Acceso en línea:http://hdl.handle.net/10261/182852
Access Level:acceso abierto
Palabra clave:Processivity
Protein engineering
Strand displacement
Helix-hairpin-helix domain
DNA amplification
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spelling Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifsVega, Miguel deLázaro, José M.Mencía, MarioBlanco Dávila, LuisSalas, MargaritaProcessivityProtein engineeringStrand displacementHelix-hairpin-helix domainDNA amplificationBacteriophage φ29 DNA polymerase is a unique enzyme endowed with two distinctive properties, high processivity and faithful polymerization coupled to strand displacement, that have led to the development of protocols to achieve isothermal amplification of limiting amounts of both circular plasmids and genomic DNA. To enhance the amplification efficiency of φ29 DNA polymerase, we have constructed chimerical DNA polymerases by fusing DNA binding domains to the C terminus of the polymerase. The results show that the addition of Helix-hairpin-Helix [(HhH)2] domains increases DNA binding of the hybrid polymerases without hindering their replication rate. In addition, the chimerical DNA polymerases display an improved and faithful multiply primed DNA amplification proficiency on both circular plasmids and genomic DNA and are unique φ29 DNA polymerase variants with enhanced amplification performance. The reported chimerical DNA polymerases will contribute to make φ29 DNA polymerase-based amplification technologies one of the most powerful tools for genomics.Autonomous Community of Madrid grant P-MAT-0283-0505 (M.S.); and by an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo OchoaPeer reviewedPeer ReviewedNational Academy of Sciences (U.S.)Fundación Ramón ArecesComunidad de MadridConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2019201920102019info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/182852reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Ingléshttps://doi.org/10.1073/pnas.1011428107Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1828522026-05-22T06:33:51Z
dc.title.none.fl_str_mv Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs
title Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs
spellingShingle Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs
Vega, Miguel de
Processivity
Protein engineering
Strand displacement
Helix-hairpin-helix domain
DNA amplification
title_short Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs
title_full Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs
title_fullStr Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs
title_full_unstemmed Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs
title_sort Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs
dc.creator.none.fl_str_mv Vega, Miguel de
Lázaro, José M.
Mencía, Mario
Blanco Dávila, Luis
Salas, Margarita
author Vega, Miguel de
author_facet Vega, Miguel de
Lázaro, José M.
Mencía, Mario
Blanco Dávila, Luis
Salas, Margarita
author_role author
author2 Lázaro, José M.
Mencía, Mario
Blanco Dávila, Luis
Salas, Margarita
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Fundación Ramón Areces
Comunidad de Madrid
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Processivity
Protein engineering
Strand displacement
Helix-hairpin-helix domain
DNA amplification
topic Processivity
Protein engineering
Strand displacement
Helix-hairpin-helix domain
DNA amplification
description Bacteriophage φ29 DNA polymerase is a unique enzyme endowed with two distinctive properties, high processivity and faithful polymerization coupled to strand displacement, that have led to the development of protocols to achieve isothermal amplification of limiting amounts of both circular plasmids and genomic DNA. To enhance the amplification efficiency of φ29 DNA polymerase, we have constructed chimerical DNA polymerases by fusing DNA binding domains to the C terminus of the polymerase. The results show that the addition of Helix-hairpin-Helix [(HhH)2] domains increases DNA binding of the hybrid polymerases without hindering their replication rate. In addition, the chimerical DNA polymerases display an improved and faithful multiply primed DNA amplification proficiency on both circular plasmids and genomic DNA and are unique φ29 DNA polymerase variants with enhanced amplification performance. The reported chimerical DNA polymerases will contribute to make φ29 DNA polymerase-based amplification technologies one of the most powerful tools for genomics.
publishDate 2010
dc.date.none.fl_str_mv 2010
2019
2019
2019
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/10261/182852
url http://hdl.handle.net/10261/182852
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv https://doi.org/10.1073/pnas.1011428107
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv National Academy of Sciences (U.S.)
publisher.none.fl_str_mv National Academy of Sciences (U.S.)
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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