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A novel cell permeable DNA replication and repair marker

Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or sh...

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Autores principales: Herce, Henry D, Rajan, Malini, Lättig-Tünnemann, Gisela, Fillies, Marion, Cardoso, M Cristina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615156/
https://www.ncbi.nlm.nih.gov/pubmed/25484186
http://dx.doi.org/10.4161/nucl.36290
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author Herce, Henry D
Rajan, Malini
Lättig-Tünnemann, Gisela
Fillies, Marion
Cardoso, M Cristina
author_facet Herce, Henry D
Rajan, Malini
Lättig-Tünnemann, Gisela
Fillies, Marion
Cardoso, M Cristina
author_sort Herce, Henry D
collection PubMed
description Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells.
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spelling pubmed-46151562015-11-03 A novel cell permeable DNA replication and repair marker Herce, Henry D Rajan, Malini Lättig-Tünnemann, Gisela Fillies, Marion Cardoso, M Cristina Nucleus Research Paper Proliferating Cell Nuclear Antigen (PCNA) is a key protein in DNA replication and repair. The dynamics of replication and repair in live cells is usually studied introducing translational fusions of PCNA. To obviate the need for transfection and bypass the problem of difficult to transfect and/or short lived cells, we have now developed a cell permeable replication and/or repair marker. The design of this marker has three essential molecular components: (1) an optimized artificial PCNA binding peptide; (2) a cell-penetrating peptide, derived from the HIV-1 Trans Activator of Transcription (TAT); (3) an in vivo cleavable linker, linking the two peptides. The resulting construct was taken up by human, hamster and mouse cells within minutes of addition to the media. Inside the cells, the cargo separated from the vector peptide and bound PCNA effectively. Both replication and repair sites could be directly labeled in live cells making it the first in vivo cell permeable peptide marker for these two fundamental cellular processes. Concurrently, we also introduced a quick peptide based PCNA staining method as an alternative to PCNA antibodies for immunofluorescence applications. In summary, we present here a versatile tool to instantaneously label repair and replication processes in fixed and live cells. Taylor & Francis 2014-10-31 /pmc/articles/PMC4615156/ /pubmed/25484186 http://dx.doi.org/10.4161/nucl.36290 Text en © 2014 The Author(s). Published with license by Taylor & Francis http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
spellingShingle Research Paper
Herce, Henry D
Rajan, Malini
Lättig-Tünnemann, Gisela
Fillies, Marion
Cardoso, M Cristina
A novel cell permeable DNA replication and repair marker
title A novel cell permeable DNA replication and repair marker
title_full A novel cell permeable DNA replication and repair marker
title_fullStr A novel cell permeable DNA replication and repair marker
title_full_unstemmed A novel cell permeable DNA replication and repair marker
title_short A novel cell permeable DNA replication and repair marker
title_sort novel cell permeable dna replication and repair marker
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615156/
https://www.ncbi.nlm.nih.gov/pubmed/25484186
http://dx.doi.org/10.4161/nucl.36290
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