Parp1 hsa

PARylated and MARylated PARP1 proteins were prepared as described 26 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, Inc., San Diego, CA, USA). Briefly, for the PAR hydrolysis activity assays, 70 nm PARP1 (PARP1‐HSA; Trevigen) was automodified as described in 26. After 20‐min incubation, PARP1 was passed three times through SpinTrap G‐25 (GE Healthcare). PARylated PARP1 substrate was used in a 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 min and analysed by NuPAGE Novex Bis‐Tris 4–12% gel using MOPS buffer (Invitrogen). Radiolabelled experiments were visualized by autoradiography.

PARP1 HSA (trevigen) was incubated in the presence or absence of A3, ABT888, or equal volume of DMSO at indicated concentrations in PARylation buffer (20 mM Tris 7.9, 100 mM NaCl, 10 mM MgCl2, 10 mM ZnCl2, 10% Glycerol, 1 mM DTT, 1 mg/ml ssDNA (Sigma), 300 uM B-NAD (Amresco)) for 30′ at 30 degrees C. Negative control reactions are performed in the absence of B-NAD. 2x SDS PAGE sample buffer stops the reaction and sample is loaded on SDS-PAGE for subsequent western-blotting for PAR and PARP1.