Visualisation of PCNA Monoubiquitination In Vivo by Single Pass Spectral Imaging FRET Microscopy

Monoubiquitination of the DNA sliding clamp, PCNA, plays a central role in the control of damage bypass during replication. By combining a widely-spaced FRET donor/acceptor pair (CFP and mRFP) with spectral imaging, we have developed a simple method for the visualisation of PCNA monoubiquitination i...

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Detalles Bibliográficos
Autores principales: Batters, Christopher, Zhu, Hannah, Sale, Julian E.
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Research Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2814862/
https://www.ncbi.nlm.nih.gov/pubmed/20126408
http://dx.doi.org/10.1371/journal.pone.0009008
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author Batters, Christopher
Zhu, Hannah
Sale, Julian E.
author_facet Batters, Christopher
Zhu, Hannah
Sale, Julian E.
author_sort Batters, Christopher
collection PubMed
description Monoubiquitination of the DNA sliding clamp, PCNA, plays a central role in the control of damage bypass during replication. By combining a widely-spaced FRET donor/acceptor pair (CFP and mRFP) with spectral imaging, we have developed a simple method for the visualisation of PCNA monoubiquitination in both fixed and live cells with a single imaging pass. We validate the method with genetic controls in the avian cell line DT40 and use it to examine the intracellular dynamics of PCNA ubiquitination following subnuclear UV irradiation. This general approach is likely to be of utility for live imaging of post-translational modifications of a wide range of substrates in vivo.
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spelling pubmed-28148622010-02-03 Visualisation of PCNA Monoubiquitination In Vivo by Single Pass Spectral Imaging FRET Microscopy Batters, Christopher Zhu, Hannah Sale, Julian E. PLoS One Research Article Monoubiquitination of the DNA sliding clamp, PCNA, plays a central role in the control of damage bypass during replication. By combining a widely-spaced FRET donor/acceptor pair (CFP and mRFP) with spectral imaging, we have developed a simple method for the visualisation of PCNA monoubiquitination in both fixed and live cells with a single imaging pass. We validate the method with genetic controls in the avian cell line DT40 and use it to examine the intracellular dynamics of PCNA ubiquitination following subnuclear UV irradiation. This general approach is likely to be of utility for live imaging of post-translational modifications of a wide range of substrates in vivo. Public Library of Science 2010-02-02 /pmc/articles/PMC2814862/ /pubmed/20126408 http://dx.doi.org/10.1371/journal.pone.0009008 Text en Batters et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Batters, Christopher
Zhu, Hannah
Sale, Julian E.
Visualisation of PCNA Monoubiquitination In Vivo by Single Pass Spectral Imaging FRET Microscopy
title Visualisation of PCNA Monoubiquitination In Vivo by Single Pass Spectral Imaging FRET Microscopy
title_full Visualisation of PCNA Monoubiquitination In Vivo by Single Pass Spectral Imaging FRET Microscopy
title_fullStr Visualisation of PCNA Monoubiquitination In Vivo by Single Pass Spectral Imaging FRET Microscopy
title_full_unstemmed Visualisation of PCNA Monoubiquitination In Vivo by Single Pass Spectral Imaging FRET Microscopy
title_short Visualisation of PCNA Monoubiquitination In Vivo by Single Pass Spectral Imaging FRET Microscopy
title_sort visualisation of pcna monoubiquitination in vivo by single pass spectral imaging fret microscopy
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2814862/
https://www.ncbi.nlm.nih.gov/pubmed/20126408
http://dx.doi.org/10.1371/journal.pone.0009008
work_keys_str_mv AT batterschristopher visualisationofpcnamonoubiquitinationinvivobysinglepassspectralimagingfretmicroscopy
AT zhuhannah visualisationofpcnamonoubiquitinationinvivobysinglepassspectralimagingfretmicroscopy
AT salejuliane visualisationofpcnamonoubiquitinationinvivobysinglepassspectralimagingfretmicroscopy

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