RNA-MEDIATED PHASE SEPARATION OF 53BP1: CONNECTING CONDENSATE BIOPHYSICS TO DNA DAMAGE RESPONSE

Biomolecular condensates, also known as membraneless organelles, are intracellular compartments which can form via liquid-liquid phase separation (LLPS) where biochemical reactions concentrate within the cell. An example of such condensates are DNA damage response (DDR) foci, proteins and RNA assemblies where DNA damage signaling and repair occur. We previously demonstrated that the DDR involves both proteins and sequence-specific RNAs and that DNA damage-induced long non-coding RNAs (dilncRNAs) and their processed shorter forms DDR RNAs (DDRNAs) facilitate the recruitment of DDR factors around DNA double-strand breaks (DSBs). Recently, we reported that among DDR components, 53BP1 accumulates at DNA damage sites in liquid-like foci, with RNA supporting this process and aiding the DNA repair pathway. However, mechanisms regulating 53BP1 condensation by RNA remain elusive. Here, we identify 53BP1 molecular determinants driving RNA-mediated LLPS. By combining computational analyses, in vitro microscopy, and binding assays, we demonstrate that the oligomerization domain (OD) and its C-terminal disordered region containing a glycine-arginine-rich (GAR) motif are essential for phase separation. Mutagenesis experiments revealed that arginine residues in the GAR motif are crucial for RNA binding and condensate formation, while lysine substitutions only partially retain these functions. Using Differential Dynamic Microscopy (DDM), FRAP, and coalescence assays, we characterize the microrheological properties of 53BP1-RNA condensates. They exhibit a two-component behavior, with a viscoelastic gel-like core surrounded by a more fluid interfacial layer. A progressive loss of fluidity of the condensates also suggests a time- dependent maturation process, transitioning toward a more solid-like state, confirmed also by FRAP in living cells. Importantly, R>K substitutions in the GAR motif not only impair RNA binding but also induce aberrant LLPS, leading to condensates with reduced internal mobility and arrested fusions, further supporting the central role of arginine residues in the physiological assembly of 53BP1 foci. Notably, we show that the GAR motif of 53BP1 is required for efficient non-homologous end joining (NHEJ) of deprotected telomeres, as measured by the frequency of telomere fusions, highlighting its functional relevance in DNA repair. Finally, we prove that antisense oligonucleotides (ASOs) targeting telomeric transcripts reduce protein-RNA binding affinity, providing a possible mechanistic explanation for ASO-induced dissolution of DDR foci. Collectively, we propose that the GAR motif of 53BP1 drives RNA interactions that are necessary for protein LLPS, and that RNA, by enriching into condensates, reshapes the composition of 53BP1 foci, creating a highly viscous liquid phase that possibly enables efficient DNA repair.