Fig. 3

N-terminus of PARP2 mediates the branched PAR chain formation. a The NTR domain of PARP2 recognizes PAR. The recombinant GST fusion proteins were incubated with ³²P labeled PAR. Protein-PAR complex was pulled down by glutathione agarose beads followed by autoradiography (top panel). Recombinant GST and GST-RNF146 WWE domain were used as the negative and positive controls (NC and PC), respectively. The GST fusion proteins were also examined by the SDS-PAGE followed with Coomassie blue staining (bottom panel). b The NTR region of PARP2 is required for the interaction with PAR. Full length PARP2, Δ NTR or Δ WGR were incubated with ³²P labeled PAR. Associated PAR was examined (top panel). Recombinant PARP2 was stained with Coomassie blue (bottom panel). c The NTR domain is required for the activation of PARP2. Biotin-NAD⁺ was used in the in vitro PARP2-dependent PARylation assays. PARP2-dependent PARylation was examined by dot blot using streptavidin-HRP. The proteins were also examined by the SDS-PAGE followed with Coomassie blue staining (bottom panel). d The levels of PARP2 in U2OS and PARP2-null cells were examined by western blot (upper panel). The PARP2-null cells were reconstituted with full length PARP2 or the Δ NTR mutant. And their expression was confirmed by western blot (lower panel). e The NTR domain is required for the branched PAR chain synthesis in response to DNA damage. Cells were treated with 500 μM H₂O₂ for 10 min. PAR was extracted and processed by PPase and AP. R₂-Ado was quantified by LC–MS/MS examination. Data are represented as mean ± s.d. as indicated from three independent experiments. Significance of differences was evaluated by Student’s t-test. NS: non significant; ***statistically significant (p < 0.001)