53BP1—DNA repair enters a new liquid phase

Abstract

In response to DNA damage, transient repair compartments in the nucleus concentrate repair proteins and activate downstream signaling factors. 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. This liquid droplet‐like behavior of 53BP1 compartments might help to coordinate local lesion recognition with global gene activation in response to DNA damage.

Phase 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). Phase separated proteins self‐organize into liquid‐like droplets, allowing certain molecules to become concentrated while excluding others. Phase separation can be driven by the formation of weak non‐covalent bonds within binding partners, and the presence of often intrinsically disordered domains that induce self‐interactions (Banani et al, 2017). The assembly of such macromolecular condensates is an emerging mechanism for achieving subcellular organization of highly dynamic, membrane‐less 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. This finding extends the list of nuclear proteins exhibiting liquid‐like behavior and suggests a role for 53BP1 liquid‐like assembly at DNA lesions as facilitator of downstream gene regulation.

In response to DNA damage, a signaling cascade orchestrated by several proteins recognizes the DNA lesions, locally amplifies the signal, and globally triggers cell cycle arrest and DNA repair. DDR factor assembly at sites of damage involves complex spatial and temporal coordination of many dynamic interactions among repair proteins and with chromatin, thereby manifesting focal assemblies called DNA repair foci (Misteli & Soutoglou, 2009). Phosphorylation of histone H2AX (γH2AX) by central DDR kinases and recruitment of the adaptor protein MDC1 at damaged sites are among the earliest discernible focal events, followed by accumulation of the scaffold protein 53BP1 (Panier & Boulton, 2014). 53BP1 generates chromatin domains surrounding DNA lesions, which are required to recruit downstream effectors, to shield the damaged DNA from extensive nucleolytic processing, and to propagate signaling to arrest the cell cycle. How this spatial and temporal confinement of protein assemblies at DNA damaged sites is achieved, and what role it plays in controlling downstream functions, remains poorly understood.

Kilic et al (2019) used state‐of‐the‐art microscopy to analyze the physicochemical properties of 53BP1 assemblies at damaged sites induced by ionizing radiation (IR) or at spontaneous 53BP1‐decorated DNA damage after replication stress in mammalian cells (so‐called 53BP1 nuclear bodies). Intriguingly, they observed that 53BP1 foci exhibit all hallmarks of phase separated compartments and exhibit droplet‐like behavior. First, 53BP1 assemblies are reversibly abolished upon increasing levels of osmotic stress, elevated temperature, or in the presence of an alcohol that disrupts weak hydrophobic interactions. Second, 53BP1 foci show droplet‐like properties, such as spherical shape and fast dynamics, and the ability to fuse together, verifying previous observations (Roukos et al, 2013). Third, experiments using an optogenetic platform, which uses light to test whether proteins undergo phase separation and form spatiotemporally definable liquid droplets (Shin et al, 2017), further supported the ability of 53BP1 to phase separate and self‐assemble. Finally, the authors confirmed the liquid‐like nature of 53BP1 assemblies by showing that also the purified protein phase‐separates in solution, and the condensates are recruited to DSB‐mimicking DNA in vitro. Intriguingly, while the initial assembly of γH2AX/MDC1 at damaged sites is essential for 53BP1 recruitment, only 53BP1 exhibits droplet‐like properties. Using live‐cell microscopy experiments to track the recruitment of both MDC1 and 53BP1 to sites of DNA damage in individual cells, Kilic et al further uncovered that fusion of 53BP1 repair compartments, a key characteristic of its droplet‐like behavior, occurs predominantly after the disappearance of MDC1 foci. This finding indicates that phase separation of 53BP1 DNA repair compartments is temporally distinct and uncoupled from the assembly of upstream repair factors.

Kilic et al (2019) further identified the sequence elements within 53BP1 that drive phase separation. They found that the carboxy‐terminal region, highly enriched in amino acids such as tyrosine and arginine, is sufficient for 53BP1 phase separation. Regions with similar composition have been found to mediate multivalent interactions promoting phase separation of several proteins with droplet‐like behavior (Banani et al, 2017). Within the sequence elements sufficient for 53BP1 clustering lies the domain that is required for auto‐oligomerization of 53BP1 and the tandem BRCT (BRCA1 C‐terminus) domain, an evolutionarily conserved protein–protein interaction module found in a diverse range of DDR proteins. These 53BP1 domains were recently shown to mediate stabilization of the tumor suppressor p53 and to promote p53 target gene expression in response to DNA damage (Cuella‐Martin et al, 2016), a finding that motivated the authors to further investigate the role of 53BP1 liquid–liquid phase separation on global p53 activation and cell cycle arrest. Intriguingly, p53 indeed accumulates at light‐induced 53BP1 droplets and at a fraction of endogenous 53BP1 nuclear bodies, indicating that 53BP1 phase separated compartments might play a role to dynamically assemble and stabilize p53 upon DNA damage. In support of this idea, the group uncovered that under conditions abrogating 53BP1 phase separation without preventing recruitment of upstream factors, the interaction between 53BP1 and p53, subsequent p53 stabilization, and expression of downstream targets that arrest the cell cycle are highly reduced. This finding suggests that 53BP1 phase separation might help coordinating local lesion recognition with global gene activation in response to DNA damage (Fig 1). However, the 53BP1 sequence elements required for phase separation and for the DDR significantly overlap, precluding analysis that could directly assess the contribution of droplet‐like 53BP1 behavior on downstream DDR functions. Similar to the effect of conditions that abrogate 53BP1 phase separation, 53BP1 deficiency led to defective checkpoint activation, suggesting that p53‐mediated effects are, at least partially, dependent on 53BP1 functions. The significance of 53BP1 phase separation on DDR deserves further attention in the near future, as it is likely that the role of phase separated 53BP1 repair compartments extends beyond activation of p53 and its downstream targets.

Figure 1. 53BP1 phase separation upon DNA damage.

The DNA damage response effector 53BP1 generates characteristic repair foci at DNA breaks. These foci exhibit liquid‐like self‐assembly that appears to be important for activation of downstream tumor suppressor p53 target genes and cell cycle arrest in response to DNA damage.

Liquid–liquid phase separation at sites of DNA damage has been previously reported (Altmeyer et al, 2015; Patel et al, 2015). Nucleated by the biopolymer poly(ADP‐ribose) (PAR), several intrinsically disordered proteins transiently accumulated at damaged sites (Altmeyer et al, 2015; Patel et al, 2015). This early assembly was shown to be incompatible with 53BP1 accumulation (Altmeyer et al, 2015), suggesting that these two distinct phase separated compartments neither temporally overlap nor intermix. Future studies are required to shed light on how local compartments seeded by different macromolecules with droplet‐like behavior interact in space and time at damaged DNA sites.

Phase separation of biomolecular condensates appears to be a central mechanism for organization of various nuclear compartments, such as the heterochromatin (Larson et al, 2017; Strom et al, 2017), the nucleolus (Feric et al, 2016), and several nuclear bodies, including PML bodies (Zhu & Brangwynne, 2015). Intriguingly, DNA break dynamics and pathway choice for repair are interlinked to the chromatin micro‐environment surrounding the lesion and to its position within the nucleus (Kalousi & Soutoglou, 2016). DNA breaks within the heterochromatin or the nucleolus, for example, move to the periphery of these compartments to complete repair, while PML bodies appear to be the favorable sites for clustering and repair of a subset of telomeres (Kalousi & Soutoglou, 2016). It is therefore possible that the different physicochemical properties of biomolecular condensates surrounding the damaged DNA and phase‐separated assemblies within various nuclear compartments may account for the observed clustering or exclusion. Combining our understanding on basic properties of phase separation and DNA repair in the context of nuclear architecture with novel methodologies that can probe multiple phase separated compartments in space and time will be an essential step in better understanding the key principles that maintain the integrity of our genome.

The EMBO Journal (2019) 38: e102871