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^WT [PAR-PARP1^WT] or its catalytic variants PARP1^Y986S and PARP1^Y986H, respectively, synthesizing (short PAR)-PARP1^Y986S or (short hyperbranched PAR)-PARP1^Y986H using dynamic light scattering, fluorescence microscopy, turbidity assays, and atomic force microscopy. We observed that biologically relevant cations such as Mg²⁺, Ca²⁺, or Mn²⁺ or polyamines (spermine⁴⁺ or spermidine³⁺) were essential for the assembly of FUS with PAR-PARP1^WT and FUS with PAR-PARP1^Y986S 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^Y986H (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.