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A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling
Preblastoderm Drosophila embryo development is characterized by fast cycles of nuclear divisions. Extracts from these embryos can be used to reconstitute complex chromatin with high efficiency. We now discovered that this chromatin assembly system contains activities that recognize unprotected DNA e...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698661/ https://www.ncbi.nlm.nih.gov/pubmed/31147711 http://dx.doi.org/10.1093/nar/gkz473 |
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author | Harpprecht, Lisa Baldi, Sandro Schauer, Tamas Schmidt, Andreas Bange, Tanja Robles, Maria S Kremmer, Elisabeth Imhof, Axel Becker, Peter B |
author_facet | Harpprecht, Lisa Baldi, Sandro Schauer, Tamas Schmidt, Andreas Bange, Tanja Robles, Maria S Kremmer, Elisabeth Imhof, Axel Becker, Peter B |
author_sort | Harpprecht, Lisa |
collection | PubMed |
description | Preblastoderm Drosophila embryo development is characterized by fast cycles of nuclear divisions. Extracts from these embryos can be used to reconstitute complex chromatin with high efficiency. We now discovered that this chromatin assembly system contains activities that recognize unprotected DNA ends and signal DNA damage through phosphorylation. DNA ends are initially bound by Ku and MRN complexes. Within minutes, the phosphorylation of H2A.V (homologous to γH2A.X) initiates from DNA breaks and spreads over tens of thousands DNA base pairs. The γH2A.V phosphorylation remains tightly associated with the damaged DNA and does not spread to undamaged DNA in the same reaction. This first observation of long-range γH2A.X spreading along damaged chromatin in an in vitro system provides a unique opportunity for mechanistic dissection. Upon further incubation, DNA ends are rendered single-stranded and bound by the RPA complex. Phosphoproteome analyses reveal damage-dependent phosphorylation of numerous DNA-end-associated proteins including Ku70, RPA2, CHRAC16, the exonuclease Rrp1 and the telomer capping complex. Phosphorylation of spindle assembly checkpoint components and of microtubule-associated proteins required for centrosome integrity suggests this cell-free system recapitulates processes involved in the regulated elimination of fatally damaged syncytial nuclei. |
format | Online Article Text |
id | pubmed-6698661 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-66986612019-08-22 A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling Harpprecht, Lisa Baldi, Sandro Schauer, Tamas Schmidt, Andreas Bange, Tanja Robles, Maria S Kremmer, Elisabeth Imhof, Axel Becker, Peter B Nucleic Acids Res Genome Integrity, Repair and Replication Preblastoderm Drosophila embryo development is characterized by fast cycles of nuclear divisions. Extracts from these embryos can be used to reconstitute complex chromatin with high efficiency. We now discovered that this chromatin assembly system contains activities that recognize unprotected DNA ends and signal DNA damage through phosphorylation. DNA ends are initially bound by Ku and MRN complexes. Within minutes, the phosphorylation of H2A.V (homologous to γH2A.X) initiates from DNA breaks and spreads over tens of thousands DNA base pairs. The γH2A.V phosphorylation remains tightly associated with the damaged DNA and does not spread to undamaged DNA in the same reaction. This first observation of long-range γH2A.X spreading along damaged chromatin in an in vitro system provides a unique opportunity for mechanistic dissection. Upon further incubation, DNA ends are rendered single-stranded and bound by the RPA complex. Phosphoproteome analyses reveal damage-dependent phosphorylation of numerous DNA-end-associated proteins including Ku70, RPA2, CHRAC16, the exonuclease Rrp1 and the telomer capping complex. Phosphorylation of spindle assembly checkpoint components and of microtubule-associated proteins required for centrosome integrity suggests this cell-free system recapitulates processes involved in the regulated elimination of fatally damaged syncytial nuclei. Oxford University Press 2019-08-22 2019-05-31 /pmc/articles/PMC6698661/ /pubmed/31147711 http://dx.doi.org/10.1093/nar/gkz473 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 Harpprecht, Lisa Baldi, Sandro Schauer, Tamas Schmidt, Andreas Bange, Tanja Robles, Maria S Kremmer, Elisabeth Imhof, Axel Becker, Peter B A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling |
title | A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling |
title_full | A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling |
title_fullStr | A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling |
title_full_unstemmed | A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling |
title_short | A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling |
title_sort | drosophila cell-free system that senses dna breaks and triggers phosphorylation signalling |
topic | Genome Integrity, Repair and Replication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698661/ https://www.ncbi.nlm.nih.gov/pubmed/31147711 http://dx.doi.org/10.1093/nar/gkz473 |
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