Fig. 7: Mono-Asp/Glu-ADPr belongs to the initial wave of PARP1 signaling.

A HPF1KO (top) or WT (bottom) U2OS cells were treated with 2 mM H₂O₂ for the indicated time points, followed by harvesting and lysis according to our optimized procedure and immunoblotted with the indicated antibodies. See also Supplementary Fig. 7 for staining with additional antibodies. Shown is a representative result from three independent experiments. B Right: immunofluorescence quantification of HPF1KO (top) or WT (bottom) U2OS cells, treated with 2 mM H₂O₂ for the indicated time points, fixed with methanol and stained with the indicated antibody and DAPI. The intensity of ~ 100 cells from a representative of 3 independent experiments is shown. Red bars represent sample mean. Left: representative confocal images. Scale bar = 10 µm. C HPF1KO (left) or WT (right) U2OS cells were treated with 1 µM PARGi or DMSO, followed by 2 mM H₂O₂ treatment at the indicated times, lysed according to our optimized procedure and immunoblotted with the indicated antibodies. Shown is a representative result from three independent experiments. D Simplified schematic of the main PARP1 signaling steps described in this study. Upon DNA damage, free PARP1 synthesizes mono-ADPr on aspartate and glutamate residues of several target proteins, including PARP1 itself. Mono-Asp/Glu-ADPr is, together with the known poly-Asp/Glu-ADPr, part of an initial wave of PARP1 signaling. Mono-Asp/Glu-ADPr is partly degraded by PARG and, consequently, PARG depletion or inhibition dramatically raises mono-Asp/Glu-ADPr, even in the presence of HPF1. By contrast, the PARP1:HPF1 complex switches catalytic activity towards formation of mono-Ser-ADPr, part of a second wave of PARP1 signaling and removed by ARH3. This schematic was generated in Adobe Illustrator using some graphical elements adapted from Longarini et al.¹⁷.