Phi 29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein

Phage phi 29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as the primer for initiation of DNA synthesis. In one of the most important motifs present in the 3'5' exonucleolytic domain of proofreading DNA po...

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Autores: Eisenbrandt, Ralf, Lázaro, José M., Salas, Margarita, Vega, Miguel de
Tipo de recurso: artículo
Fecha de publicación:2002
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/7832
Acceso en línea:http://hdl.handle.net/10261/7832
Access Level:acceso abierto
Palabra clave:Phage phi 29
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spelling Phi 29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal proteinEisenbrandt, RalfLázaro, José M.Salas, MargaritaVega, Miguel dePhage phi 29Phage phi 29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as the primer for initiation of DNA synthesis. In one of the most important motifs present in the 3'5' exonucleolytic domain of proofreading DNA polymerases, the ExoII motif, phi 29 DNA polymerase contains three amino acid residues, Y59, H61 and F69, which are highly conserved among most proofreading DNA polymerases. These residues have recently been shown to be involved in proper stabilization of the primer terminus at the 3'5' exonuclease active site. Here we investigate by means of site-directed mutagenesis the role of these three residues in reactions that are specific for DNA polymerases utilizing a protein-primed DNA replication mechanism. Mutations introduced at residues Y59, H61 and F69 severely affected the protein-primed replication capacity of phi 29 DNA polymerase. For four of the mutants, namely Y59L, H61L, H61R and F69S, interaction with the terminal protein was affected, leading to few initiation and transition products. These findings, together with the specific conservation of Y59, H61 and F69 among DNA polymerases belonging to the protein-primed subgroup, strongly suggest a functional role of these amino acid residues in the DNA polymerase–terminal protein interactionThis investigation was aided by research grant 5R01 GM27242-22 from the National Institutes of Health, by grant PB98-0645 from the Dirección General de Investigacíon Científica y Técnica, by grant ERBFMX CT97 0125 from the European Union and by an institutional grant from Fundación Ramón Areces. R.E. was a post-doctoral fellow of the European UnionPeer reviewedOxford University PressNational Institutes of Health (US)Ministerio de Economía y Competitividad (España)European CommissionFundación Ramón Areces200820082002info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501291941 bytesapplication/pdfhttp://hdl.handle.net/10261/7832reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Ingléshttp://nar.oxfordjournals.org/cgi/content/full/30/6/1379info:eu-repo/semantics/openAccessoai:digital.csic.es:10261/78322026-05-22T06:33:51Z
dc.title.none.fl_str_mv Phi 29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein
title Phi 29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein
spellingShingle Phi 29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein
Eisenbrandt, Ralf
Phage phi 29
title_short Phi 29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein
title_full Phi 29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein
title_fullStr Phi 29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein
title_full_unstemmed Phi 29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein
title_sort Phi 29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein
dc.creator.none.fl_str_mv Eisenbrandt, Ralf
Lázaro, José M.
Salas, Margarita
Vega, Miguel de
author Eisenbrandt, Ralf
author_facet Eisenbrandt, Ralf
Lázaro, José M.
Salas, Margarita
Vega, Miguel de
author_role author
author2 Lázaro, José M.
Salas, Margarita
Vega, Miguel de
author2_role author
author
author
dc.contributor.none.fl_str_mv National Institutes of Health (US)
Ministerio de Economía y Competitividad (España)
European Commission
Fundación Ramón Areces
dc.subject.none.fl_str_mv Phage phi 29
topic Phage phi 29
description Phage phi 29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as the primer for initiation of DNA synthesis. In one of the most important motifs present in the 3'5' exonucleolytic domain of proofreading DNA polymerases, the ExoII motif, phi 29 DNA polymerase contains three amino acid residues, Y59, H61 and F69, which are highly conserved among most proofreading DNA polymerases. These residues have recently been shown to be involved in proper stabilization of the primer terminus at the 3'5' exonuclease active site. Here we investigate by means of site-directed mutagenesis the role of these three residues in reactions that are specific for DNA polymerases utilizing a protein-primed DNA replication mechanism. Mutations introduced at residues Y59, H61 and F69 severely affected the protein-primed replication capacity of phi 29 DNA polymerase. For four of the mutants, namely Y59L, H61L, H61R and F69S, interaction with the terminal protein was affected, leading to few initiation and transition products. These findings, together with the specific conservation of Y59, H61 and F69 among DNA polymerases belonging to the protein-primed subgroup, strongly suggest a functional role of these amino acid residues in the DNA polymerase–terminal protein interaction
publishDate 2002
dc.date.none.fl_str_mv 2002
2008
2008
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/10261/7832
url http://hdl.handle.net/10261/7832
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv http://nar.oxfordjournals.org/cgi/content/full/30/6/1379
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 291941 bytes
application/pdf
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
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