Fig. 1

PAR biosensors detect cellular PARylation. a PAR-binding biosensors derived from APLF and CHFR. The PAR-binding PBZ domains were fused to full-length GFP, generating PAR biosensors PBZ-GFP and CHFR-PBZ-GFP, respectively. When CHFR-PBZ biosensor is used it is explicitly annotated; in all other cases the APLF PBZ biosensor is used. b, c PAR-binding biosensor signal correlates with PAR immunodetection but has a greater dynamic range. HeLa cells expressing PBZ-GFP were exposed to 1 mM H₂O₂ or 1 μM olaparib; GFP signal and PAR (10 H anti-PAR antibody) were monitored 10 min after exposure. b Scatter plots of the PAR biosensor intensity correlate with the PAR Ab detection. Intensities were measure in > 1000 cells per condition. c Fold change in median signal in H₂O₂ vs. mock, and olaparib vs. mock are shown for both PAR detection approaches (exemplary images are shown in Supplementary Fig. 1A). NS–not significant, box plot shows quartiles, Student’s t-test **p-values < 0.01. d, f Kinetics of PARylation at sites of DNA damage. PARP1 null CAL51 cells (CAL51 PARP1^–/–) were transfected with PARP1-GFP and PBZ-mRuby2 and exposed to localized (micro)irradiation as shown in d. After microirradiation, PARP1 and PAR localization were monitored over time. A microirradiated cell is shown; the area that is shown on the subsequent kymographs is annotated with a white box with a 2 µm side. Scale bar represents 5 µm. e Kymograph (top) and graph (bottom) of PARP1 and PBZ-mRuby2 0–3 s after microirradiation. f as per e but 0–10 min after microirradiation. Each graph shows average signals from > 10 cells; scale bar represents a distance of 2 µm. g, h A PAR biosensor detects loss of PAR at microirradiated sites caused by PARP1 mutations. g PARP1 bound to a double strand break (4OQB) with indicated: p.[43delM;44 F > I] and E988K mutations in red. h Kymographs are shown from microirradiated CAL51 PARP1^–/– cells expressing PBZ-mRuby2 and either wild-type PARP1-GFP, PARP1-p.[43delM;44 F > I]-GFP, or PARP1-E988K-GFP. i The clinical PARP inhibitor talazoparib reduces PAR levels at sites of microirradiation. Kymographs of HeLa cells with a PARP1-GFP containing bacterial artificial chromosome (PARP1-LAP) and PBZ-mRuby2 were exposed to 100 nM talazoparib for 1 h prior to microirradiation