Fig. 1: HPF1 regulation of PARP1 and PARP2.

a The catalytic domains of PARP1 (4DQY), PARP2 (4PJV), and PARP3 (3FHB) are superimposed. The helical domain (HD) and the ADP-ribosyltransferase (ART) domain are labeled. The Donor ADPr site is defined by the PARP1 complex with BAD (6BHV), and the Acceptor ADPr site is defined by the PARP1 complex with carba-NAD⁺ (1A26). PARP1 and PARP2 share a similar structure of the loop that bears residue H826 (PARP1), whereas PARP3 has a truncated loop (*). PARP1 and PARP2 share conserved residues at their C-terminal ends that are not conserved in PARP3 (C-termini labeled “C”). b The ART of PARP2 (HD deleted) in complex with HPF1 (6TX3). A conserved His residue from the loop highlighted in panel B (826 in PARP1), and a Trp residue at the C-terminus (1014 in PARP1), are drawn as sticks and labeled (His and Trp). The Donor site is bound by the NAD⁺ mimic EB47 that was crystallized with the complex. HPF1 binding clashes with the Acceptor site (carba-NAD⁺ is shown bound to the acceptor site as in panel B in order to highlight the clash). HPF1 residue E284 directly contributes to the PARP active site. c PARP1 or PARP2 (1 μM) was incubated with dumbbell DNA containing a central nick (1 μM) with or without HPF1 (1 μM) for 10 min at room temperature (RT). 500 μM NAD⁺ was added for 5 min and reactions were quenched with 500 µM PARP inhibitor (olaparib or talazoparib). Where indicated, reactions were treated with 1 M hydroxylamine (NH₂OH) for one hour. Reactions were resolved by SDS-PAGE and treated with Imperial Stain. d Same as panel c for PARP1 WT, mutant H826E, and mutant ΔCterm (Δ1012–1014), with 0.1 μM of HPF1. The experiment in c was performed three times. The experiment in d was performed two 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.