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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...

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Autores principales: Harpprecht, Lisa, Baldi, Sandro, Schauer, Tamas, Schmidt, Andreas, Bange, Tanja, Robles, Maria S, Kremmer, Elisabeth, Imhof, Axel, Becker, Peter B
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
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.
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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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