SUMMARY- PROJECT 3-PARP: PAR-dependent, phase-transitioned protein assemblies and DNA repair Poly-ADP-ribose polymerases (PARPs) utilize NAD+ as a donor for ADP-ribose modification of proteins and for the subsequent additions that generate poly-ADP-ribose (PAR) on these targets. PARP1 and PARP2 are the predominant enzymes in humans that catalyze PARylation in response to DNA damage and are critical targets of cancer therapeutics. Rapid formation of PAR occurs at many different types of DNA lesions and has been shown to be is required for the timely recruitment of DNA repair enzymes. In addition, PAR recruits polypeptides containing low-complexity domains and intrinsically disordered regions. Some of these disordered proteins form phase transitions between soluble, gel, and fibrous states. PAR thus seeds phase condensates at sites of DNA damage to create a specialized compartment for repairing DNA. Despite the fundamental importance of this phenomenon, we know very little about the structural basis for PARP generation of PAR polymers at DNA damage sites or the mechanistic role of PAR-seeded phase transitions in promoting DNA repair. Here we hypothesize that PAR-driven phase separation regulates DNA repair outcomes at diverse DNA lesions and that the length and structure of PAR polymer is critical for tuning these outcomes. We will test this hypothesis by investigating the mechanistic basis of ADP-ribose addition and PAR formation by PARP1/2 in the context of cofactor HPF1 using structural biology, ensemble biochemistry, and single-molecule methods (Aim 1). Structural insights from this effort will produce a working model for PARP1/2 catalytic activity as well as a toolbox of PARP1/2 mutants that generate varying lengths and structures of PAR. As part of this effort we will also develop inhibitors for the primary glycohydrolase that removes DNA damage-induced ADP-ribose modifications (ARH3). We will probe the role of phase transitions in DNA repair by using FUS and other low-complexity proteins to investigate the structural basis of their interactions with PAR-seeded domains of various lengths (Aim 2). Lastly, we will determine the effects of phase transitions on the recruitment and activity of DNA double-strand break repair complexes in vitro, as well as the effects of these transitions on the repair of multiple types of DNA damage in human cells (Aim 3). These efforts join P3 with the other projects in SBDR5 by collaborating to identify effects of lesion-specific PARylation on the repair of alkylation damage, double-strand breaks, and stalled replication forks. In addition, we will determine the composition of PAR-seeded domains in response to DNA damage and how this changes in response to clinically used PARP and glycohydrolase inhibitors. This project will produce structural and mechanistic insights into the dynamic regulation of PAR synthesis and disassembly that occurs in human cells and will determine how the PAR-seeded phase transitions at ...