Phase Separation of FUS with Poly(ADP-ribosyl)ated PARP1 Is Controlled by Polyamines, Divalent Metal Cations, and Poly(ADP-ribose) Structure.

Fused in sarcoma (FUS) is involved in the formation of nuclear biomolecular condensates associated with poly(ADP-ribose) [PAR] synthesis catalyzed by a DNA damage sensor such as PARP1. Here, we studied FUS microphase separation induced by poly(ADP-ribosyl)ated PARP1 [PAR-PARP1] or its catalytic variants PARP1 and PARP1, respectively, synthesizing (short PAR)-PARP1 or (short hyperbranched PAR)-PARP1 using dynamic light scattering, fluorescence microscopy, turbidity assays, and atomic force microscopy. We observed that biologically relevant cations such as Mg, Ca, or Mn or polyamines (spermine or spermidine) were essential for the assembly of FUS with PAR-PARP1 and FUS with PAR-PARP1 in vitro. We estimated the range of the FUS-to-PAR-PARP1 molar ratio and the cation concentration that are favorable for the stability of the protein's microphase-separated state. We also found that FUS microphase separation induced by PAR-PARP1 (i.e., a PARP1 variant attaching short hyperbranched PAR to itself) can occur in the absence of cations. The dependence of PAR-PARP1-induced FUS microphase separation on cations and on the branching of the PAR structure points to a potential role of the latter in the regulation of the formation of FUS-related biological condensates and requires further investigation.