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A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling.
Nucleic Acids Res. 47, 7444-7459 (2019)
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 gamma H2A.X) initiates from DNA breaks and spreads over tens of thousands DNA base pairs. The gamma H2A.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 gamma H2A.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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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
Double-strand Breaks; Chromatin Accessibility; Xenopus-laevis; Messenger-rna; Repair; Complex; Protein; Damage; Dynamics; Melanogaster
Language
english
Publication Year
2019
HGF-reported in Year
2019
ISSN (print) / ISBN
0305-1048
e-ISSN
1362-4962
Journal
Nucleic Acids Research
Quellenangaben
Volume: 47,
Issue: 14,
Pages: 7444-7459
Publisher
Oxford University Press
Publishing Place
Great Clarendon St, Oxford Ox2 6dp, England
Reviewing status
Peer reviewed
Institute(s)
Institute of Molecular Immunology (IMI)
POF-Topic(s)
30504 - Mechanisms of Genetic and Environmental Influences on Health and Disease
Research field(s)
Immune Response and Infection
PSP Element(s)
G-501793-001
WOS ID
WOS:000483815100028
Scopus ID
85071702691
Erfassungsdatum
2019-09-24