SUMMARY Poly(ADP-ribose) polymerase-1 (PARP-1) creates the posttranslational modification PAR from substrate NAD+ to regulate multiple cellular processes. DNA breaks sharply elevate PARP-1 catalytic activity to mount a cell survival repair response, whereas persistent PARP-1 hyperactivation during severe genotoxic stress is associated with cell death. The mechanism for tight control of the robust catalytic potential of PARP-1 remains unclear. By monitoring PARP-1 dynamics using hydrogen/deuterium exchange-mass spectrometry (HXMS), we unexpectedly find that a specific portion of the helical subdomain (HD) of the catalytic domain rapidly unfolds when PARP-1 encounters a DNA break. Together with biochemical and crystallographic analysis of HD deletion mutants, we show that the HD is an autoinhibitory domain that blocks productive NAD+ binding. Our molecular model explains how PARP-1 DNA damage detection leads to local unfolding of the HD that relieves autoinhibition, and has important implications for the design of PARP inhibitors.

PARP-1, PARP-2 and PARP-3 are DNA-dependent PARPs that localize to DNA damage, synthesize poly(ADP-ribose) (PAR) covalently attached to target proteins including themselves, and thereby recruit repair factors to DNA breaks to increase repair efficiency. PARP-1, PARP-2 and PARP-3 have in common two C-terminal domains—Trp-Gly-Arg (WGR) and catalytic (CAT). In contrast, the N-terminal region (NTR) of PARP-1 is over 500 residues and includes four regulatory domains, whereas PARP-2 and PARP-3 have smaller NTRs (70 and 40 residues, respectively) of unknown structural composition and function. Here, we show that PARP-2 and PARP-3 are preferentially activated by DNA breaks harboring a 5′ phosphate (5′P), suggesting selective activation in response to specific DNA repair intermediates, in particular structures that are competent for DNA ligation. In contrast to PARP-1, the NTRs of PARP-2 and PARP-3 are not strictly required for DNA binding or for DNA-dependent activation. Rather, the WGR domain is the central regulatory domain of PARP-2 and PARP-3. Finally, PARP-1, PARP-2 and PARP-3 share an allosteric regulatory mechanism of DNA-dependent catalytic activation through a local destabilization of the CAT. Collectively, our study provides new insights into the specialization of the DNA-dependent PARPs and their specific roles in DNA repair pathways.

SummaryPoly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1’s function remained obscure; inherent dynamics of SSBs and PARP-1’s multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1’s signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodifcation in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins.