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Phase separation of 53 BP 1 determines liquid‐like behavior of DNA repair compartments


Kilic, Sinan; Lezaja, Aleksandra; Gatti, Marco; Bianco, Eliana; Michelena, Jone; Imhof, Ralph; Altmeyer, Matthias (2019). Phase separation of 53 BP 1 determines liquid‐like behavior of DNA repair compartments. EMBO Journal Online, 38(16):e101379.

Abstract

The DNA damage response (DDR) generates transient repair compartments to concentrate repair proteins and activate signaling factors. The physicochemical properties of these spatially confined compartments and their function remain poorly understood. Here, we establish, based on live cell microscopy and CRISPR/Cas9-mediated endogenous protein tagging, that 53BP1-marked repair compartments are dynamic, show droplet-like behavior, and undergo frequent fusion and fission events. 53BP1 assembly, but not the upstream accumulation of γH2AX and MDC1, is highly sensitive to changes in osmotic pressure, temperature, salt concentration and to disruption of hydrophobic interactions. Phase separation of 53BP1 is substantiated by optoDroplet experiments, which further allowed dissection of the 53BP1 sequence elements that cooperate for light-induced clustering. Moreover, we found the tumor suppressor protein p53 to be enriched within 53BP1 optoDroplets, and conditions that disrupt 53BP1 phase separation impair 53BP1-dependent induction of p53 and diminish p53 target gene expression. We thus suggest that 53BP1 phase separation integrates localized DNA damage recognition and repair factor assembly with global p53-dependent gene activation and cell fate decisions.

Abstract

The DNA damage response (DDR) generates transient repair compartments to concentrate repair proteins and activate signaling factors. The physicochemical properties of these spatially confined compartments and their function remain poorly understood. Here, we establish, based on live cell microscopy and CRISPR/Cas9-mediated endogenous protein tagging, that 53BP1-marked repair compartments are dynamic, show droplet-like behavior, and undergo frequent fusion and fission events. 53BP1 assembly, but not the upstream accumulation of γH2AX and MDC1, is highly sensitive to changes in osmotic pressure, temperature, salt concentration and to disruption of hydrophobic interactions. Phase separation of 53BP1 is substantiated by optoDroplet experiments, which further allowed dissection of the 53BP1 sequence elements that cooperate for light-induced clustering. Moreover, we found the tumor suppressor protein p53 to be enriched within 53BP1 optoDroplets, and conditions that disrupt 53BP1 phase separation impair 53BP1-dependent induction of p53 and diminish p53 target gene expression. We thus suggest that 53BP1 phase separation integrates localized DNA damage recognition and repair factor assembly with global p53-dependent gene activation and cell fate decisions.

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Item Type:Journal Article, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Department of Molecular Mechanisms of Disease
07 Faculty of Science > Department of Molecular Mechanisms of Disease
Dewey Decimal Classification:570 Life sciences; biology
Scopus Subject Areas:Life Sciences > General Neuroscience
Life Sciences > Molecular Biology
Life Sciences > General Biochemistry, Genetics and Molecular Biology
Life Sciences > General Immunology and Microbiology
Uncontrolled Keywords:General Biochemistry, Genetics and Molecular Biology, General Immunology and Microbiology, General Neuroscience, Molecular Biology
Language:English
Date:1 July 2019
Deposited On:26 Aug 2019 16:35
Last Modified:09 May 2021 07:29
Publisher:Nature Publishing Group
ISSN:0261-4189
OA Status:Hybrid
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.15252/embj.2018101379
PubMed ID:31267591
Project Information:
  • : FunderSNSF
  • : Grant IDPP00P3_179057
  • : Project TitleUncovering concealed regulators and unexpected pathway connections guarding against genome instability
  • : FunderSNSF
  • : Grant IDPP00P3_150690
  • : Project TitleUncovering concealed regulators and unexpected pathway connections guarding against genome instability
  • : FunderH2020
  • : Grant ID714326
  • : Project TitleDiVineGenoMe - Decoding cell-to-cell variation in genome integrity maintenance

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