Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path.

Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3'-5' exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerizatio...

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Detalles Bibliográficos
Autores: Dodd, Thomas, Botto, Margherita, Paul, Fabian, Lamers, Meindert H, Ivanov, Ivaylo, Fernandez-Leiro, Rafael
Tipo de recurso: artículo
Fecha de publicación:2020
País:España
Institución:Instituto de Salud Carlos III (ISCIII)
Repositorio:Repisalud
Idioma:inglés
OAI Identifier:oai:repisalud.isciii.es:20.500.12105/12182
Acceso en línea:http://hdl.handle.net/20.500.12105/12182
Access Level:acceso abierto
Palabra clave:Polymerization
DNA
DNA Polymerase III
DNA Primers
DNA Replication
DNA-Directed DNA Polymerase
Escherichia coli
Exonucleases
Kinetics
Models, Molecular
Protein Conformation
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spelling Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path.Dodd, ThomasBotto, MargheritaPaul, FabianLamers, Meindert HIvanov, IvayloFernandez-Leiro, RafaelDodd, ThomasBotto, MargheritaPaul, FabianLamers, Meindert HIvanov, IvayloFernandez-Leiro, RafaelPolymerizationDNADNA Polymerase IIIDNA PrimersDNA ReplicationDNA-Directed DNA PolymeraseEscherichia coliExonucleasesKineticsModels, MolecularProtein ConformationProofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3'-5' exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of E. coli replicative DNA polymerase Pol III. We identify structures for all intermediates, in which the transitioning primer strand is stabilized by conserved Pol III residues along the fingers, thumb and exonuclease domains. We demonstrate switching kinetics on a tens of milliseconds timescale and unveil a complete pol-to-exo switching mechanism, validated by targeted mutational experiments.Nature Publishing GroupUnited States Department of Health and Human ServicesNational Science Foundation (Estados Unidos)United States Department of Energy20212021-03-1020202020-11-0120202020-11-01journal articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfapplication/pdfapplication/pdfvideo/quicktimeapplication/pdfhttp://hdl.handle.net/20.500.12105/12182reponame:Repisaludinstname:Instituto de Salud Carlos III (ISCIII)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2Atribución-NoComercial-CompartirIgual 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc-sa/4.0/info:eu-repo/semantics/openAccessoai:repisalud.isciii.es:20.500.12105/121822026-06-12T12:43:37Z
dc.title.none.fl_str_mv Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path.
title Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path.
spellingShingle Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path.
Dodd, Thomas
Polymerization
DNA
DNA Polymerase III
DNA Primers
DNA Replication
DNA-Directed DNA Polymerase
Escherichia coli
Exonucleases
Kinetics
Models, Molecular
Protein Conformation
title_short Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path.
title_full Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path.
title_fullStr Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path.
title_full_unstemmed Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path.
title_sort Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path.
dc.creator.none.fl_str_mv Dodd, Thomas
Botto, Margherita
Paul, Fabian
Lamers, Meindert H
Ivanov, Ivaylo
Fernandez-Leiro, Rafael
Dodd, Thomas
Botto, Margherita
Paul, Fabian
Lamers, Meindert H
Ivanov, Ivaylo
Fernandez-Leiro, Rafael
author Dodd, Thomas
author_facet Dodd, Thomas
Botto, Margherita
Paul, Fabian
Lamers, Meindert H
Ivanov, Ivaylo
Fernandez-Leiro, Rafael
author_role author
author2 Botto, Margherita
Paul, Fabian
Lamers, Meindert H
Ivanov, Ivaylo
Fernandez-Leiro, Rafael
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv United States Department of Health and Human Services
National Science Foundation (Estados Unidos)
United States Department of Energy
dc.subject.none.fl_str_mv Polymerization
DNA
DNA Polymerase III
DNA Primers
DNA Replication
DNA-Directed DNA Polymerase
Escherichia coli
Exonucleases
Kinetics
Models, Molecular
Protein Conformation
topic Polymerization
DNA
DNA Polymerase III
DNA Primers
DNA Replication
DNA-Directed DNA Polymerase
Escherichia coli
Exonucleases
Kinetics
Models, Molecular
Protein Conformation
description Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3'-5' exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of E. coli replicative DNA polymerase Pol III. We identify structures for all intermediates, in which the transitioning primer strand is stabilized by conserved Pol III residues along the fingers, thumb and exonuclease domains. We demonstrate switching kinetics on a tens of milliseconds timescale and unveil a complete pol-to-exo switching mechanism, validated by targeted mutational experiments.
publishDate 2020
dc.date.none.fl_str_mv 2020
2020-11-01
2020
2020-11-01
2021
2021-03-10
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12105/12182
url http://hdl.handle.net/20.500.12105/12182
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
Atribución-NoComercial-CompartirIgual 4.0 Internacional
http://creativecommons.org/licenses/by-nc-sa/4.0/
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
Atribución-NoComercial-CompartirIgual 4.0 Internacional
http://creativecommons.org/licenses/by-nc-sa/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
video/quicktime
application/pdf
dc.publisher.none.fl_str_mv Nature Publishing Group
publisher.none.fl_str_mv Nature Publishing Group
dc.source.none.fl_str_mv reponame:Repisalud
instname:Instituto de Salud Carlos III (ISCIII)
instname_str Instituto de Salud Carlos III (ISCIII)
reponame_str Repisalud
collection Repisalud
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,811543

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