<span class="paragraphSection"><div class="boxTitle">Abstract</div>FUS participates in the formation of biomolecular condensates associated with PARP1-dependent synthesis of poly(ADP-ribose) (PAR). HPF1 regulates auto- and hetero-PARylation activities of PARP1 and PARP2 and may influence the formation of FUS compartments during PARP1 or PARP2 auto-PARylation. In this study, we used atomic force microscopy in combination with biochemical assay to investigate the formation of FUS compartments under activation of PARP1 and PARP2, when HPF1 modulates their activity. Similar to PARP1, FUS and PARylated PARP2 form DNA-rich compartments, indicating that PARP2 PARylation is sufficient for the formation of such compartments. The excess of HPF1 over PARP1 diminishes PARP1 activity and reduces the size of DNA-rich compartments. However, an excess of HPF1 over PARP2 does not significantly affect PARP2 activity and the size of compartments. Furthermore, HPF1 stimulates hetero-PARylation of FUS; this modification is stronger with PARP2 than with PARP1. HPF1-dependent intensive PARylation of FUS catalyzed by PARP1 or PARP2 impairs the assembly of DNA-rich compartment. These data provide a basis for investigating the effect of HPF1 on the formation of PAR-dependent condensates involving RNA-binding proteins like FUS, which interact effectively with PAR and show the ability to be targets of PARylation to regulate condensate formation at DNA damage sites.</span>