Fig. 2. HPF1 works efficiently at sub-stochiometric ratios relative to PARP1 and PARP2.

a PARP1 (1 μM) or b PARP2 (1 μM) was incubated with various amounts of HPF1 at the ratios indicated in the presence of DNA (1 μM) for 10 min at RT. Five-hundred micromolar NAD⁺ was added for 5 min and reactions were quenched with 500 μM PARPi (olaparib or talazoparib). Where indicated, reactions were treated with hydroxylamine (NH₂OH) for 1 h. Reactions were resolved by SDS-PAGE and treated with Imperial Stain. c PARP1 (1 μM) was mixed with HPF1 (1 μM) at various time points relative to NAD⁺ addition (500 μM). For the −15’ reaction, HPF1 was incubated with PARP1 for 15 min prior to NAD⁺ addition. For the 0 reaction, HPF1 was added at the same time as NAD⁺. For the + reactions, HPF1 was added after NAD⁺ at the time indicated (20 s, 1 min, 3 min). For “post inh”, HPF1 was added after the reaction was quenched with PARP inhibitor. Reactions were then treated as in panels a and b. Experiments in a, b, and c were performed three times. Numbers on the left side of the gels represent molecular weight marker locations (values in kDa). Source data are provided as a Source Data file.