TY - JOUR

T1 - Phase separation of 53BP1 determines liquid-like behavior of DNA repair compartments

AU - Kilic, Sinan

AU - Gatti, Marco

AU - Imhof, Ralph

N1 - Funding Information: We acknowledge ETH ScopeM and UZH ZMB for excellent microscopy support. We are grateful to R. Greenberg for U-2 OS FokI cells, L. Toledo for U-2 OS GFP-53BP1 cells, Manuel Stucki for U-2 OS GFP-MDC1 cells, Jiri Lukas for the GFP-MDC1 construct, R. Chapman for MCF7 wild-type and 53BP1 KO cells, and K. Gari for FAM-labeled dsDNA. We thank Feng Zhang, Dorus Gadella, Chandra Tucker, and Titia de Lange for providing plasmids via Addgene and acknowledge all members of our lab and of the DMMD for helpful discussions. Research funding was provided by the Swiss National Science Foundation (grants 150690 and 179057), the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant 714326), the Novartis Foundation for Medical-Biological Research (grant 16B078), and the Swiss Cancer Research Foundation (grant KFS-4406-02-2018). Funding Information: We acknowledge ETH ScopeM and UZH ZMB for excellent microscopy support. We are grateful to R. Greenberg for U-2 OS FokI cells, L. Toledo for U-2 OS GFP-53BP1 cells, Manuel Stucki for U-2 OS GFP-MDC1 cells, Jiri Lukas for the GFP-MDC1 construct, R. Chapman for MCF7 wild-type and 53BP1 KO cells, and K. Gari for FAM-labeled dsDNA. We thank Feng Zhang, Dorus Gadella, Chandra Tucker, and Titia de Lange for providing plasmids via Addgene and acknowledge all members of our lab and of the DMMD for helpful discussions. Research funding was provided by the Swiss National Science Foundation (grants 150690 and 179057), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant 714326), the Novartis Foundation for Medical-Biological Research (grant 16B078), and the Swiss Cancer Research Foundation (grant KFS-4406-02-2018). Publisher Copyright: © 2019 The Authors. Published under the terms of the CC BY NC ND 4.0 license

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

KW - 53BP1

KW - DNA damage response

KW - genome stability

KW - liquid–liquid phase separation

KW - p53

UR - http://www.scopus.com/inward/record.url?scp=85068469757&partnerID=8YFLogxK

U2 - 10.15252/embj.2018101379

DO - 10.15252/embj.2018101379

M3 - Journal article

C2 - 31267591

AN - SCOPUS:85068469757

VL - 38

JO - E M B O Journal

JF - E M B O Journal

SN - 0261-4189

IS - 16

M1 - e101379

ER -