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PARP1 and PARP2 recognize DNA breaks immediately upon their formation, generate a burst of local PARylation to signal their location, and are co-targeted by all current FDA-approved forms of PARP inhibitors (PARPi) used in the cancer clinic. Recent evidence indicates that the same PARPi molecules impact PARP2 differently from PARP1, raising the possibility that allosteric activation may also differ. We find that unlike for PARP1, destabilization of the autoinhibitory domain of PARP2 is insufficient for DNA damage-induced catalytic activation. Rather, PARP2 activation requires further unfolding of an active site α-helix absent in PARP1. Only one clinical PARPi, Olaparib, stabilizes the PARP2 active site α-helix, representing a structural feature with the potential to discriminate small molecule inhibitors. Collectively, our findings reveal unanticipated differences in local structure and changes in activation-coupled backbone dynamics between PARP1 and PARP2., Competing Interests: Declarations of interest B.E.B., J.M.P., and T.T.T. are co-founders of Hysplex, Inc. with interests in PARP inhibitor development. B.E.B., J.M.P., and T.T.T. are co-inventors on a provisional patent application filed by UPenn that is related to this work. B.E.B is on the scientific advisory board of Denovicon Therapeutics.

PARP1 and PARP2 produce poly(ADP-ribose) in response to DNA breaks. HPF1 regulates PARP1/2 catalytic output, most notably permitting serine modification with ADP-ribose. However, PARP1 is substantially more abundant in cells than HPF1, challenging whether HPF1 can pervasively modulate PARP1. Here, we show biochemically that HPF1 efficiently regulates PARP1/2 catalytic output at sub-stoichiometric ratios matching their relative cellular abundances. HPF1 rapidly associates/dissociates from multiple PARP1 molecules, initiating serine modification before modification initiates on glutamate/aspartate, and accelerating initiation to be more comparable to elongation reactions forming poly(ADP-ribose). This "hit and run" mechanism ensures HPF1 contributions to PARP1/2 during initiation do not persist and interfere with PAR chain elongation. We provide structural insights into HPF1/PARP1 assembled on a DNA break, and assess HPF1 impact on PARP1 retention on DNA. Our data support the prevalence of serine-ADP-ribose modification in cells and the efficiency of serine-ADP-ribose modification required for an acute DNA damage response., (© 2021. The Author(s).)