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. Display omitted •HXMS reveals dramatic changes in PARP-1 dynamics upon binding to DNA damage•The HD subdomain locally unfolds when PARP-1 is bound to DNA damage•The HD is an autoinhibitory domain that blocks productive binding to NAD+•X-ray structures of constitutively active PARPs are determined bound to inhibitors Dawicki-McKenna, Langelier et al. identify the HD autoinhibitory domain in PARP-1 and reveal that DNA break sensing leads to local unfolding of HD, which allows proper active site positioning of NAD+ and leads to a burst in poly(ADP-ribose) production in response to DNA damage.