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

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.

53BP1—DNA repair enters a new liquid phase

“…In this issue of The EMBO Journal, Kilic et al show that DNA repair focal assemblies marked by accumulation of 53BP1 are phase separated liquid compartments. In this issue, Kilic et al (2019) find that repair compartments marked by accumulation of 53BP1, a major player in the DNA damage response (DDR), phase separate with characteristic droplet-like behavior. P hase separation of macromolecules has recently emerged as a key player in the control of many biological pathways including RNA metabolism, ribosome biogenesis, and signal transduction (Banani et al, 2017).…”

Biomolecular condensates in neurodegeneration and cancer

“…Amyloid‐like beta‐sheets also contribute to disordered region interactions . Importantly, the cooperation between oligomerization domains and disordered regions lowers the concentration threshold at which phase separation occurs, as does the interaction between proteins and other molecules such as nucleic acids (also see Table ) . Nucleic acids alone can undergo phase separation through base pairing in vitro and in cells, potentially nucleating the assembly of RNA‐protein condensates.…”

“…Loss of 53BP1 results in genome instability and radiation sensitivity in mice and correlates with rapid cancer progression and poor prognosis (reviewed in Reference ). 53BP1 phase‐separates in cells and in vitro to form dynamic condensates, which concentrate downstream factors, p53 and its coactivator USP28. Disrupting 53BP1 condensates reduces the expression of a p53 target gene, p21, and impairs p21 function of cell cycle checkpoint activation .…”

“…53BP1 phase‐separates in cells and in vitro to form dynamic condensates, which concentrate downstream factors, p53 and its coactivator USP28. Disrupting 53BP1 condensates reduces the expression of a p53 target gene, p21, and impairs p21 function of cell cycle checkpoint activation . Curiously, 53BP1 is excluded from the FET condensates (Figure C) and appears as foci later in the DNA repair process, indicating that these are likely distinct condensates.…”

“…Disrupting 53BP1 condensates reduces the expression of a p53 target gene, p21, and impairs p21 function of cell cycle checkpoint activation . Curiously, 53BP1 is excluded from the FET condensates (Figure C) and appears as foci later in the DNA repair process, indicating that these are likely distinct condensates. It is not yet clear whether FET and 53BP1 condensates represent different stages of an evolving condensate (ie, some components of the condensates are shared although FET and 53BP1 proteins change) or are separate condensates that assemble one after another with distinct composition and function.…”