Fig. 5
Enzyme activity of DrPARG. a Michaelis–Menten plots of wild-type (WT), T267R, T267K, and E112A DrPARG catalyzing poly-ADP-ribose (PAR) hydrolysis are shown. Initial reaction velocity (μmol/min/mg protein) is plotted against PAR substrate concentration (μM). Data are plotted as mean ± SEM (n = 3 independent experiments). WT, T267R, T267K, and E112A DrPARG are indicated by open circle, triangle, square, and diamond, respectively. b Detection of PAR in the cleavage products of DrPARG. The reaction of automodified PARP1 treated with or without (time = 0) WT and mutant DrPARG was stopped at different times and filtered with a 30-kDa cut-off column. PAR was assayed in the filtrate by dot blotting. PARGs from Homo sapiens (HsPARG) and Thermomonospora curvata (TcPARG) were used as controls of canonical and bacterial PARG, respectively. c The products of reactions treated with WT and mutant DrPARG for 15 min were filtered with a 10-kDa cut-off column and subjected to high-performance liquid chromatography (HPLC) analyses. ADP-ribose (ADPR) and residual NAD+ were indicated. The fraction indicated by an asterisk from cleavage products of WT DrPARG was collected and analyzed by mass spectrometry. d Identification of endo-glycohydrolase cleavage products of DrPARG by time-of-flight mass spectrometry (TOF MS). The cleavage products separated by HPLC were subjected to a quadrupole time-of-flight (Q-TOF) instrument for analysis. Ion chromatogram was shown with m/z ratios for deprotonated ADPR dimer (ADPR2; m/z 1099.0), doubly charged deprotonated ADPR trimer (ADPR3; m/z 819.5), and doubly charged deprotonated ADPR tetramer (ADPR4; m/z 1090.0) indicated. e Enzyme activity of DrPARG toward endogenous PAR of D. radiodurans was assayed in cell lysates from the Δparg strain treated with or without (time = 0) WT or E122A DrPARG. PAR level was assayed by dot blotting over a time course. Source data are provided as a Source Data file
