Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA

Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of...

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Autores: Lancey, Claudia, Tehseen, Muhammad, Bakshi, Souvika, Percival, Matthew, Takahashi, Masateru, Sobhy, Mohamed A., Raducanu, Vlad S., Blair, Kerry, Muskett, Frederick W., Ragan, Timothy J., Crehuet, Ramón, Hamdan, Samir M., De Biasio, Alfredo
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:10261/254063
Acceso en línea:http://hdl.handle.net/10261/254063
Access Level:acceso abierto
Palabra clave:Cryoelectron microscopy
DNA
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spelling Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNALancey, ClaudiaTehseen, MuhammadBakshi, SouvikaPercival, MatthewTakahashi, MasateruSobhy, Mohamed A.Raducanu, Vlad S.Blair, KerryMuskett, Frederick W.Ragan, Timothy J.Crehuet, RamónHamdan, Samir M.De Biasio, AlfredoCryoelectron microscopyDNAY-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.This research was supported by King Abdullah University of Science and Technology through core funding (to S.M.H.) and the Competitive Research Award Grant CRG8 URF/1/4036‐01‐01 (to S.M.H. and A.D.B.), and by the Wellcome Trust (to A.D.B.). R.C. acknowledges funding from the MINECO (CTQ2016-78636-P) and to AGAUR, (2017 SGR 324). The MD project has been carried out using CSUC resources. We acknowledge The Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology (LISCB), major funding from MRC (MC_PC_17136). We thank Christos Savva (LISCB, University of Leicester) for his help in cryo-EM data collection and advice on data processing.Peer reviewedNature Publishing GroupMinisterio de Economía y Competitividad (España)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202120212021info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/254063reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/CTQ2016-78636-Phttps://doi.org/10.1038/s41467-021-26251-6Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/2540632026-05-22T06:33:51Z
dc.title.none.fl_str_mv Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA
title Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA
spellingShingle Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA
Lancey, Claudia
Cryoelectron microscopy
DNA
title_short Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA
title_full Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA
title_fullStr Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA
title_full_unstemmed Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA
title_sort Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA
dc.creator.none.fl_str_mv Lancey, Claudia
Tehseen, Muhammad
Bakshi, Souvika
Percival, Matthew
Takahashi, Masateru
Sobhy, Mohamed A.
Raducanu, Vlad S.
Blair, Kerry
Muskett, Frederick W.
Ragan, Timothy J.
Crehuet, Ramón
Hamdan, Samir M.
De Biasio, Alfredo
author Lancey, Claudia
author_facet Lancey, Claudia
Tehseen, Muhammad
Bakshi, Souvika
Percival, Matthew
Takahashi, Masateru
Sobhy, Mohamed A.
Raducanu, Vlad S.
Blair, Kerry
Muskett, Frederick W.
Ragan, Timothy J.
Crehuet, Ramón
Hamdan, Samir M.
De Biasio, Alfredo
author_role author
author2 Tehseen, Muhammad
Bakshi, Souvika
Percival, Matthew
Takahashi, Masateru
Sobhy, Mohamed A.
Raducanu, Vlad S.
Blair, Kerry
Muskett, Frederick W.
Ragan, Timothy J.
Crehuet, Ramón
Hamdan, Samir M.
De Biasio, Alfredo
author2_role author
author
author
author
author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Economía y Competitividad (España)
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Cryoelectron microscopy
DNA
topic Cryoelectron microscopy
DNA
description Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.
publishDate 2021
dc.date.none.fl_str_mv 2021
2021
2021
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/10261/254063
url http://hdl.handle.net/10261/254063
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/CTQ2016-78636-P
https://doi.org/10.1038/s41467-021-26251-6
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Nature Publishing Group
publisher.none.fl_str_mv Nature Publishing Group
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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