Enzymatic Hydrolysis of PARylated and MARylated PARP1

PARylated and MARylated PARP1 proteins were prepared as described [26 (link)] in a reaction buffer containing 50 mM Tris-HCl (pH 8.0), 4 mM MgCl₂, 50 mM NaCl, 0.2 mM DTT, 200 μM NAD+ (Trevigen) and 130 ng activated DNA (BPS Bioscience). Briefly, for the PAR hydrolysis activity assays, 70 nM PARP1 (PARP1-HSA; Trevigen) was auto-modified as described in [26 (link)]. After 20 min incubation, of PARP1 was passed three times through SpinTrap G-25 (GE Healthcare). PARylated PARP1 substrate was used in 10 μL reaction. For the MARylated PARP1, 1 μM of PARP1-E988Q was used as a substrate. Reactions were stopped by the addition of PARP inhibitor Olaparib (1 μM). The MgCl₂ (Sigma) concentration was adjusted to 15 mM to allow full hydrolase activity. Automodified PARP1 was then incubated for indicated times at 30°C with hydrolytic enzymes in 10 μL reaction. Concentrations of hydrolytic enzymes used are as indicated in figures. Reactions were stopped by addition of Laemmli loading buffer, samples boiled at 90°C for 1.5 minutes and analysed by NuPAGE Novex Bis-Tris 4–12% Gel using MOPS buffer (Invitrogen). Radiolabelled experiments were visualized by autoradiography.