Cargando…
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...
Autores principales: | , , , , , |
---|---|
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 |
_version_ | 1783411400855519232 |
---|---|
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. |
format | Online Article Text |
id | pubmed-6468286 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT steurerbarbara fluorescentlylabelledcpdand64ppphotolyasesnewtoolsforlivecelldnadamagequantificationandlaserassistedrepair AT turkyilmazyasemin fluorescentlylabelledcpdand64ppphotolyasesnewtoolsforlivecelldnadamagequantificationandlaserassistedrepair AT vantoornmarvin fluorescentlylabelledcpdand64ppphotolyasesnewtoolsforlivecelldnadamagequantificationandlaserassistedrepair AT vanleeuwenwessel fluorescentlylabelledcpdand64ppphotolyasesnewtoolsforlivecelldnadamagequantificationandlaserassistedrepair AT escuderoferruzpaula fluorescentlylabelledcpdand64ppphotolyasesnewtoolsforlivecelldnadamagequantificationandlaserassistedrepair AT marteijnjurgena fluorescentlylabelledcpdand64ppphotolyasesnewtoolsforlivecelldnadamagequantificationandlaserassistedrepair |