Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes

In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks at natural or exogenous obstacles causes "replicative stress" that promotes genomic instability and affects cellular fit...

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Detalhes bibliográficos
Autores: Rodriguez-Acebes, Sara, Mouron, Silvana Andrea, Mendez, Juan
Formato: artículo
Fecha de publicación:2018
País:España
Recursos:Instituto de Salud Carlos III (ISCIII)
Repositorio:Repisalud
Idioma:inglés
OAI Identifier:oai:repisalud.isciii.es:20.500.12105/8366
Acesso em linha:http://hdl.handle.net/20.500.12105/8366
Access Level:acceso abierto
Palavra-chave:Aphidicolin
Cell Cycle Proteins
Cellular Senescence
DNA
DNA Repair
DNA Replication
HeLa Cells
Humans
Protein-Serine-Threonine Kinases
Replication Origin
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spelling Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypesRodriguez-Acebes, SaraMouron, Silvana AndreaMendez, JuanAphidicolinCell Cycle ProteinsCellular SenescenceDNADNA RepairDNA ReplicationHeLa CellsHumansProtein-Serine-Threonine KinasesReplication OriginIn growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks at natural or exogenous obstacles causes "replicative stress" that promotes genomic instability and affects cellular fitness. Replicative stress phenotypes can be characterized at the single-molecule level with DNA combing or stretched DNA fibers, but interpreting the results obtained with these approaches is complicated by the fact that the speed of replication forks is connected to the frequency of origin activation. Primary alterations in fork speed trigger secondary responses in origins, and, conversely, primary alterations in the number of active origins induce compensatory changes in fork speed. Here, by employing interventions that temporally restrict either fork speed or origin firing while still allowing interrogation of the other variable, we report a set of experimental conditions to separate cause and effect in any manipulation that affects DNA replication dynamics. Using HeLa cells and chemical inhibition of origin activity (through a CDC7 kinase inhibitor) and of DNA synthesis (via the DNA polymerase inhibitor aphidicolin), we found that primary effects of replicative stress on velocity of replisomes (fork rate) can be readily distinguished from primary effects on origin firing. Identifying the primary cause of replicative stress in each case as demonstrated here may facilitate the design of methods to counteract replication stress in primary cells or to enhance it in cancer cells to increase their susceptibility to therapies that target DNA repair.American Society for Biochemistry and Molecular Biology (ASBMB)Ministerio de Economía y Competitividad (España)20192019-09-2420182018-08-1720182018-08-17journal articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/20.500.12105/8366reponame:Repisaludinstname:Instituto de Salud Carlos III (ISCIII)InglésengES BFU2013-49153-P Not availableES CSD2007-00015 Not availableES BFU2016-80402-R Not availableopen 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:20.500.12105/83662026-06-12T12:43:37Z
dc.title.none.fl_str_mv Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes
title Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes
spellingShingle Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes
Rodriguez-Acebes, Sara
Aphidicolin
Cell Cycle Proteins
Cellular Senescence
DNA
DNA Repair
DNA Replication
HeLa Cells
Humans
Protein-Serine-Threonine Kinases
Replication Origin
title_short Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes
title_full Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes
title_fullStr Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes
title_full_unstemmed Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes
title_sort Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes
dc.creator.none.fl_str_mv Rodriguez-Acebes, Sara
Mouron, Silvana Andrea
Mendez, Juan
author Rodriguez-Acebes, Sara
author_facet Rodriguez-Acebes, Sara
Mouron, Silvana Andrea
Mendez, Juan
author_role author
author2 Mouron, Silvana Andrea
Mendez, Juan
author2_role author
author
dc.contributor.none.fl_str_mv Ministerio de Economía y Competitividad (España)
dc.subject.none.fl_str_mv Aphidicolin
Cell Cycle Proteins
Cellular Senescence
DNA
DNA Repair
DNA Replication
HeLa Cells
Humans
Protein-Serine-Threonine Kinases
Replication Origin
topic Aphidicolin
Cell Cycle Proteins
Cellular Senescence
DNA
DNA Repair
DNA Replication
HeLa Cells
Humans
Protein-Serine-Threonine Kinases
Replication Origin
description In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks at natural or exogenous obstacles causes "replicative stress" that promotes genomic instability and affects cellular fitness. Replicative stress phenotypes can be characterized at the single-molecule level with DNA combing or stretched DNA fibers, but interpreting the results obtained with these approaches is complicated by the fact that the speed of replication forks is connected to the frequency of origin activation. Primary alterations in fork speed trigger secondary responses in origins, and, conversely, primary alterations in the number of active origins induce compensatory changes in fork speed. Here, by employing interventions that temporally restrict either fork speed or origin firing while still allowing interrogation of the other variable, we report a set of experimental conditions to separate cause and effect in any manipulation that affects DNA replication dynamics. Using HeLa cells and chemical inhibition of origin activity (through a CDC7 kinase inhibitor) and of DNA synthesis (via the DNA polymerase inhibitor aphidicolin), we found that primary effects of replicative stress on velocity of replisomes (fork rate) can be readily distinguished from primary effects on origin firing. Identifying the primary cause of replicative stress in each case as demonstrated here may facilitate the design of methods to counteract replication stress in primary cells or to enhance it in cancer cells to increase their susceptibility to therapies that target DNA repair.
publishDate 2018
dc.date.none.fl_str_mv 2018
2018-08-17
2018
2018-08-17
2019
2019-09-24
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/20.500.12105/8366
url http://hdl.handle.net/20.500.12105/8366
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.relation.none.fl_str_mv ES BFU2013-49153-P Not available
ES CSD2007-00015 Not available
ES BFU2016-80402-R Not available
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
dc.publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology (ASBMB)
publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology (ASBMB)
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 15,812429

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