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Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair

UV light induces cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs), which can result in carcinogenesis and aging, if not properly repaired by nucleotide excision repair (NER). Assays to determine DNA damage load and repair rates are invaluable tools for fund...

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Autores principales: Steurer, Barbara, Turkyilmaz, Yasemin, van Toorn, Marvin, van Leeuwen, Wessel, Escudero-Ferruz, Paula, Marteijn, Jurgen A
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468286/
https://www.ncbi.nlm.nih.gov/pubmed/30698791
http://dx.doi.org/10.1093/nar/gkz035
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author Steurer, Barbara
Turkyilmaz, Yasemin
van Toorn, Marvin
van Leeuwen, Wessel
Escudero-Ferruz, Paula
Marteijn, Jurgen A
author_facet Steurer, Barbara
Turkyilmaz, Yasemin
van Toorn, Marvin
van Leeuwen, Wessel
Escudero-Ferruz, Paula
Marteijn, Jurgen A
author_sort Steurer, Barbara
collection PubMed
description UV light induces cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs), which can result in carcinogenesis and aging, if not properly repaired by nucleotide excision repair (NER). Assays to determine DNA damage load and repair rates are invaluable tools for fundamental and clinical NER research. However, most current assays to quantify DNA damage and repair cannot be performed in real time. To overcome this limitation, we made use of the damage recognition characteristics of CPD and 6-4PP photolyases (PLs). Fluorescently-tagged PLs efficiently recognize UV-induced DNA damage without blocking NER activity, and therefore can be used as sensitive live-cell damage sensors. Importantly, FRAP-based assays showed that PLs bind to damaged DNA in a highly sensitive and dose-dependent manner, and can be used to quantify DNA damage load and to determine repair kinetics in real time. Additionally, PLs can instantly reverse DNA damage by 405 nm laser-assisted photo-reactivation during live-cell imaging, opening new possibilities to study lesion-specific NER dynamics and cellular responses to damage removal. Our results show that fluorescently-tagged PLs can be used as a versatile tool to sense, quantify and repair DNA damage, and to study NER kinetics and UV-induced DNA damage response in living cells.
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spelling pubmed-64682862019-04-22 Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair Steurer, Barbara Turkyilmaz, Yasemin van Toorn, Marvin van Leeuwen, Wessel Escudero-Ferruz, Paula Marteijn, Jurgen A Nucleic Acids Res Genome Integrity, Repair and Replication UV light induces cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs), which can result in carcinogenesis and aging, if not properly repaired by nucleotide excision repair (NER). Assays to determine DNA damage load and repair rates are invaluable tools for fundamental and clinical NER research. However, most current assays to quantify DNA damage and repair cannot be performed in real time. To overcome this limitation, we made use of the damage recognition characteristics of CPD and 6-4PP photolyases (PLs). Fluorescently-tagged PLs efficiently recognize UV-induced DNA damage without blocking NER activity, and therefore can be used as sensitive live-cell damage sensors. Importantly, FRAP-based assays showed that PLs bind to damaged DNA in a highly sensitive and dose-dependent manner, and can be used to quantify DNA damage load and to determine repair kinetics in real time. Additionally, PLs can instantly reverse DNA damage by 405 nm laser-assisted photo-reactivation during live-cell imaging, opening new possibilities to study lesion-specific NER dynamics and cellular responses to damage removal. Our results show that fluorescently-tagged PLs can be used as a versatile tool to sense, quantify and repair DNA damage, and to study NER kinetics and UV-induced DNA damage response in living cells. Oxford University Press 2019-04-23 2019-01-30 /pmc/articles/PMC6468286/ /pubmed/30698791 http://dx.doi.org/10.1093/nar/gkz035 Text en © The Author(s) 2019. 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 Non-Commercial 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
Steurer, Barbara
Turkyilmaz, Yasemin
van Toorn, Marvin
van Leeuwen, Wessel
Escudero-Ferruz, Paula
Marteijn, Jurgen A
Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair
title Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair
title_full Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair
title_fullStr Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair
title_full_unstemmed Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair
title_short Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair
title_sort fluorescently-labelled cpd and 6-4pp photolyases: new tools for live-cell dna damage quantification and laser-assisted repair
topic Genome Integrity, Repair and Replication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468286/
https://www.ncbi.nlm.nih.gov/pubmed/30698791
http://dx.doi.org/10.1093/nar/gkz035
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