FUS-dependent liquid-liquid phase separation is an early event in double-strand break repair

Abstract

RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). Implicated in RNA metabolism and DNA repair, the RBP Fused-in-sarcoma (FUS) contains a prion-like domain (PLD) and undergoes reversible phase separation. Here, we report that liquid-liquid phase separation (LLPS) occurs at DNA damage foci and is necessary for the efficient recruitment of key DDR factors. We show that FUS co-purifies with the DDR factor KU and with SFPQ, another PLD-containing RBP implicated in DNA repair.Moreover, we demonstrate that FUS is required for 53BP1 localisation to DNA damage foci and for the correct recruitment of KU80 and NBS1 to sites of DNA damage. LLPS-deficient FUS variants impair retention of the DNA damage sensor KU at sites of DNA damage, and the recruitment of SFPQ. These findings provide a mechanistic function for FUS-dependent LLPS in the activation of the DDR and in the recruitment of DDR factors and RBPs at sites of DNA damage.

Fused in Sarcoma (FUS) in DNA Repair: Tango with Poly(ADP-ribose) Polymerase 1 and Compartmentalisation of Damaged DNA

“…Lately, a number of fundamental discoveries were made concerning the functions of RBPs in the DDR; they may profoundly change the concept of the regulation and organisation of DNA repair processes in the cell. Notably, the recent finding that RBPs with prion-like domains undergo phase separation points to the participation of RBPs in the formation of DNA damage–induced compartments [ 17 , 19 , 56 , 101 , 104 ]. In this regard, the interaction of RBPs with the PAR produced in response to DNA damage is currently receiving increasing attention because PAR not only contributes to the recruitment of RBPs to a DNA repair site but also promotes their LLPS.…”

“…Consequently, FUS may take part in DNA strand break repair in a PAR-dependent manner, although little is known about the functional significance of FUS–PAR interactions in DNA repair [ 40 , 104 ]. One supposition is the unusual capacity of FUS to form a liquid-like compartment making it an ideal organiser of DNA repair compartments [ 17 , 101 , 104 ] able to concentrate damaged DNA at the early stages of the DNA strand break response in order to undergo spatially controlled PAR-dependent phase separation [ 101 , 104 ].…”

Is there a role of phase partitioning in coordinating DNA damage response?

“…This perspective is supported by the mounting evidence accounting for R-loops as physiological regulators in the genome (21,22). Notably, Rad52 (in yeast) and FUS (in human) were observed to contribute to the formation of a molecular biocondensate at DSB sites, carrying out different roles, namely the organization of nuclear microtubule filaments (23), protecting the resected end of lesions and promoting DNAdamage signaling (24,25), as well as recruiting other DSB-repair-related enzymes (26). Analogously, 53BP1, which is known to be important for DSB signaling and to affect the progression of cell cycle, was demonstrated to take part in liquid compartments, too (24,25).…”

Complex Chromatin Motions for DNA Repair

“…These approaches are very powerful, but also limited to the site targeted by the endonuclease. Given that repair responses and relocalization pathways are affected by chromatin state ( Delabaere and Chiolo, 2016 ; Hauer and Gasser, 2017 ), nuclear positioning ( Lemaitre et al, 2014 ), and the phase separated nature of nuclear subdomains ( Altmeyer et al, 2015 ; Kilic et al, 2019 ; Lenzken et al, 2019 ; Oshidari et al, 2019a ; Pessina et al, 2019 ) (reviewed in Clouaire and Legube, 2019 ; Rawal et al, 2019 ), the endonuclease-based approach would need to be applied to a large number of sites to provide a comprehensive understanding of the mechanisms responsible for these dynamics in different contexts.…”