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Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells
Topoisomerase 1 (Top1) is essential for removing the DNA supercoiling generated during replication and transcription. Anticancer drugs like camptothecin (CPT) and its clinical derivatives exert their cytotoxicity by reversibly trapping Top1 in covalent complexes on the DNA (Top1cc). Poly(ADP-ribose)...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5041477/ https://www.ncbi.nlm.nih.gov/pubmed/27466387 http://dx.doi.org/10.1093/nar/gkw665 |
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author | Das, Subhendu K. Rehman, Ishita Ghosh, Arijit Sengupta, Souvik Majumdar, Papiya Jana, Biman Das, Benu Brata |
author_facet | Das, Subhendu K. Rehman, Ishita Ghosh, Arijit Sengupta, Souvik Majumdar, Papiya Jana, Biman Das, Benu Brata |
author_sort | Das, Subhendu K. |
collection | PubMed |
description | Topoisomerase 1 (Top1) is essential for removing the DNA supercoiling generated during replication and transcription. Anticancer drugs like camptothecin (CPT) and its clinical derivatives exert their cytotoxicity by reversibly trapping Top1 in covalent complexes on the DNA (Top1cc). Poly(ADP-ribose) polymerase (PARP) catalyses the addition of ADP-ribose polymers (PAR) onto itself and Top1. PARP inhibitors enhance the cytotoxicity of CPT in the clinical trials. However, the molecular mechanism by which PARylation regulates Top1 nuclear dynamics is not fully understood. Using live-cell imaging of enhanced green fluorescence tagged-human Top1, we show that PARP inhibitors (Veliparib, ABT-888) delocalize Top1 from the nucleolus to the nucleoplasm, which is independent of Top1–PARP1 interaction. Using fluorescence recovery after photobleaching and subsequent fitting of the data employing kinetic modelling we demonstrate that ABT-888 markedly increase CPT-induced bound/immobile fraction of Top1 (Top1cc) across the nuclear genome, suggesting Top1-PARylation counteracts CPT-induced stabilization of Top1cc. We further show Trp205 and Asn722 of Top1 are critical for subnuclear dynamics. Top1 mutant (N722S) was restricted to the nucleolus in the presence of CPT due to its deficiency in the accumulation of CPT-induced Top1-PARylation and Top1cc formation. This work identifies ADP-ribose polymers as key determinant for regulating Top1 subnuclear dynamics. |
format | Online Article Text |
id | pubmed-5041477 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-50414772016-09-30 Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells Das, Subhendu K. Rehman, Ishita Ghosh, Arijit Sengupta, Souvik Majumdar, Papiya Jana, Biman Das, Benu Brata Nucleic Acids Res Nucleic Acid Enzymes Topoisomerase 1 (Top1) is essential for removing the DNA supercoiling generated during replication and transcription. Anticancer drugs like camptothecin (CPT) and its clinical derivatives exert their cytotoxicity by reversibly trapping Top1 in covalent complexes on the DNA (Top1cc). Poly(ADP-ribose) polymerase (PARP) catalyses the addition of ADP-ribose polymers (PAR) onto itself and Top1. PARP inhibitors enhance the cytotoxicity of CPT in the clinical trials. However, the molecular mechanism by which PARylation regulates Top1 nuclear dynamics is not fully understood. Using live-cell imaging of enhanced green fluorescence tagged-human Top1, we show that PARP inhibitors (Veliparib, ABT-888) delocalize Top1 from the nucleolus to the nucleoplasm, which is independent of Top1–PARP1 interaction. Using fluorescence recovery after photobleaching and subsequent fitting of the data employing kinetic modelling we demonstrate that ABT-888 markedly increase CPT-induced bound/immobile fraction of Top1 (Top1cc) across the nuclear genome, suggesting Top1-PARylation counteracts CPT-induced stabilization of Top1cc. We further show Trp205 and Asn722 of Top1 are critical for subnuclear dynamics. Top1 mutant (N722S) was restricted to the nucleolus in the presence of CPT due to its deficiency in the accumulation of CPT-induced Top1-PARylation and Top1cc formation. This work identifies ADP-ribose polymers as key determinant for regulating Top1 subnuclear dynamics. Oxford University Press 2016-09-30 2016-07-27 /pmc/articles/PMC5041477/ /pubmed/27466387 http://dx.doi.org/10.1093/nar/gkw665 Text en © The Author(s) 2016. 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 | Nucleic Acid Enzymes Das, Subhendu K. Rehman, Ishita Ghosh, Arijit Sengupta, Souvik Majumdar, Papiya Jana, Biman Das, Benu Brata Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells |
title | Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells |
title_full | Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells |
title_fullStr | Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells |
title_full_unstemmed | Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells |
title_short | Poly(ADP-ribose) polymers regulate DNA topoisomerase I (Top1) nuclear dynamics and camptothecin sensitivity in living cells |
title_sort | poly(adp-ribose) polymers regulate dna topoisomerase i (top1) nuclear dynamics and camptothecin sensitivity in living cells |
topic | Nucleic Acid Enzymes |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5041477/ https://www.ncbi.nlm.nih.gov/pubmed/27466387 http://dx.doi.org/10.1093/nar/gkw665 |
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