Cargando…

Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway

We present evidence that Tim establishes a physical and functional interaction with DDX11, a super-family 2 iron-sulfur cluster DNA helicase genetically linked to the chromosomal instability disorder Warsaw breakage syndrome. Tim stimulates DDX11 unwinding activity on forked DNA substrates up to 10-...

Descripción completa

Detalles Bibliográficos
Autores principales: Calì, Federica, Bharti, Sanjay Kumar, Perna, Roberta Di, Brosh, Robert M., Pisani, Francesca M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4737141/
https://www.ncbi.nlm.nih.gov/pubmed/26503245
http://dx.doi.org/10.1093/nar/gkv1112
_version_ 1782413425612161024
author Calì, Federica
Bharti, Sanjay Kumar
Perna, Roberta Di
Brosh, Robert M.
Pisani, Francesca M.
author_facet Calì, Federica
Bharti, Sanjay Kumar
Perna, Roberta Di
Brosh, Robert M.
Pisani, Francesca M.
author_sort Calì, Federica
collection PubMed
description We present evidence that Tim establishes a physical and functional interaction with DDX11, a super-family 2 iron-sulfur cluster DNA helicase genetically linked to the chromosomal instability disorder Warsaw breakage syndrome. Tim stimulates DDX11 unwinding activity on forked DNA substrates up to 10-fold and on bimolecular anti-parallel G-quadruplex DNA structures and three-stranded D-loop approximately 4–5-fold. Electrophoretic mobility shift assays revealed that Tim enhances DDX11 binding to DNA, suggesting that the observed stimulation derives from an improved ability of DDX11 to interact with the nucleic acid substrate. Surface plasmon resonance measurements indicate that DDX11 directly interacts with Tim. DNA fiber track assays with HeLa cells exposed to hydroxyurea demonstrated that Tim or DDX11 depletion significantly reduced replication fork progression compared to control cells; whereas no additive effect was observed by co-depletion of both proteins. Moreover, Tim and DDX11 are epistatic in promoting efficient resumption of stalled DNA replication forks in hydroxyurea-treated cells. This is consistent with the finding that association of the two endogenous proteins in the cell extract chromatin fraction is considerably increased following hydroxyurea exposure. Overall, our studies provide evidence that Tim and DDX11 physically and functionally interact and act in concert to preserve replication fork progression in perturbed conditions.
format Online
Article
Text
id pubmed-4737141
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-47371412016-02-03 Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway Calì, Federica Bharti, Sanjay Kumar Perna, Roberta Di Brosh, Robert M. Pisani, Francesca M. Nucleic Acids Res Genome Integrity, Repair and Replication We present evidence that Tim establishes a physical and functional interaction with DDX11, a super-family 2 iron-sulfur cluster DNA helicase genetically linked to the chromosomal instability disorder Warsaw breakage syndrome. Tim stimulates DDX11 unwinding activity on forked DNA substrates up to 10-fold and on bimolecular anti-parallel G-quadruplex DNA structures and three-stranded D-loop approximately 4–5-fold. Electrophoretic mobility shift assays revealed that Tim enhances DDX11 binding to DNA, suggesting that the observed stimulation derives from an improved ability of DDX11 to interact with the nucleic acid substrate. Surface plasmon resonance measurements indicate that DDX11 directly interacts with Tim. DNA fiber track assays with HeLa cells exposed to hydroxyurea demonstrated that Tim or DDX11 depletion significantly reduced replication fork progression compared to control cells; whereas no additive effect was observed by co-depletion of both proteins. Moreover, Tim and DDX11 are epistatic in promoting efficient resumption of stalled DNA replication forks in hydroxyurea-treated cells. This is consistent with the finding that association of the two endogenous proteins in the cell extract chromatin fraction is considerably increased following hydroxyurea exposure. Overall, our studies provide evidence that Tim and DDX11 physically and functionally interact and act in concert to preserve replication fork progression in perturbed conditions. Oxford University Press 2016-01-29 2015-10-25 /pmc/articles/PMC4737141/ /pubmed/26503245 http://dx.doi.org/10.1093/nar/gkv1112 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Genome Integrity, Repair and Replication
Calì, Federica
Bharti, Sanjay Kumar
Perna, Roberta Di
Brosh, Robert M.
Pisani, Francesca M.
Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
title Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
title_full Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
title_fullStr Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
title_full_unstemmed Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
title_short Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway
title_sort tim/timeless, a member of the replication fork protection complex, operates with the warsaw breakage syndrome dna helicase ddx11 in the same fork recovery pathway
topic Genome Integrity, Repair and Replication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4737141/
https://www.ncbi.nlm.nih.gov/pubmed/26503245
http://dx.doi.org/10.1093/nar/gkv1112
work_keys_str_mv AT califederica timtimelessamemberofthereplicationforkprotectioncomplexoperateswiththewarsawbreakagesyndromednahelicaseddx11inthesameforkrecoverypathway
AT bhartisanjaykumar timtimelessamemberofthereplicationforkprotectioncomplexoperateswiththewarsawbreakagesyndromednahelicaseddx11inthesameforkrecoverypathway
AT pernarobertadi timtimelessamemberofthereplicationforkprotectioncomplexoperateswiththewarsawbreakagesyndromednahelicaseddx11inthesameforkrecoverypathway
AT broshrobertm timtimelessamemberofthereplicationforkprotectioncomplexoperateswiththewarsawbreakagesyndromednahelicaseddx11inthesameforkrecoverypathway
AT pisanifrancescam timtimelessamemberofthereplicationforkprotectioncomplexoperateswiththewarsawbreakagesyndromednahelicaseddx11inthesameforkrecoverypathway