Human primpol discrimination against dideoxynucleotides during primer synthesis

PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replica...

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Detalles Bibliográficos
Autores: Carvalho, Gustavo, Díaz-Talavera, Alberto, Calvo, Patricia A., Blanco Dávila, Luis, Martínez-Jiménez, María I.
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2021
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/265694
Acceso en línea:http://hdl.handle.net/10261/265694
Access Level:acceso abierto
Palabra clave:PrimPol
DNA primase
Polymerase
Dideoxynucleotides
CTNA
NRTIs
Anti-retroviral
ddC
Zalcitabine
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spelling Human primpol discrimination against dideoxynucleotides during primer synthesisCarvalho, GustavoDíaz-Talavera, AlbertoCalvo, Patricia A.Blanco Dávila, LuisMartínez-Jiménez, María I.PrimPolDNA primasePolymeraseDideoxynucleotidesCTNANRTIsAnti-retroviralddCZalcitabinePrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting de novo synthesis of DNA primers, and especially when selecting the 3 nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs versus ddNTPs at the 3 elongation site, thus avoiding synthesis of abortive primers.BFU2015-65880-P (MINECO) and PGC2018-093576-B.C21 (MCI/AEI/FEDER, UE) to L.B., and by institutional grants from Fundación Ramón Areces and Banco de Santander to the Centro de Biología Molecular Severo OchoaMultidisciplinary Digital Publishing InstituteMinisterio de Economía y Competitividad (España)Fundación Ramón ArecesBanco SantanderConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2022202220212022info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/265694reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Ingléshttp://dx.doi.org/10.3390/genes12101487Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/2656942026-05-22T06:33:51Z
dc.title.none.fl_str_mv Human primpol discrimination against dideoxynucleotides during primer synthesis
title Human primpol discrimination against dideoxynucleotides during primer synthesis
spellingShingle Human primpol discrimination against dideoxynucleotides during primer synthesis
Carvalho, Gustavo
PrimPol
DNA primase
Polymerase
Dideoxynucleotides
CTNA
NRTIs
Anti-retroviral
ddC
Zalcitabine
title_short Human primpol discrimination against dideoxynucleotides during primer synthesis
title_full Human primpol discrimination against dideoxynucleotides during primer synthesis
title_fullStr Human primpol discrimination against dideoxynucleotides during primer synthesis
title_full_unstemmed Human primpol discrimination against dideoxynucleotides during primer synthesis
title_sort Human primpol discrimination against dideoxynucleotides during primer synthesis
dc.creator.none.fl_str_mv Carvalho, Gustavo
Díaz-Talavera, Alberto
Calvo, Patricia A.
Blanco Dávila, Luis
Martínez-Jiménez, María I.
author Carvalho, Gustavo
author_facet Carvalho, Gustavo
Díaz-Talavera, Alberto
Calvo, Patricia A.
Blanco Dávila, Luis
Martínez-Jiménez, María I.
author_role author
author2 Díaz-Talavera, Alberto
Calvo, Patricia A.
Blanco Dávila, Luis
Martínez-Jiménez, María I.
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Economía y Competitividad (España)
Fundación Ramón Areces
Banco Santander
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv PrimPol
DNA primase
Polymerase
Dideoxynucleotides
CTNA
NRTIs
Anti-retroviral
ddC
Zalcitabine
topic PrimPol
DNA primase
Polymerase
Dideoxynucleotides
CTNA
NRTIs
Anti-retroviral
ddC
Zalcitabine
description PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting de novo synthesis of DNA primers, and especially when selecting the 3 nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs versus ddNTPs at the 3 elongation site, thus avoiding synthesis of abortive primers.
publishDate 2021
dc.date.none.fl_str_mv 2021
2022
2022
2022
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/10261/265694
url http://hdl.handle.net/10261/265694
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv http://dx.doi.org/10.3390/genes12101487
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Multidisciplinary Digital Publishing Institute
publisher.none.fl_str_mv Multidisciplinary Digital Publishing Institute
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 15,811543

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