Preparation and Hydrolysis of PARylated and MARylated PARP1

Poly(ADP-ribosyl)ated and mono(ADP-ribosylated) PARP1 proteins were prepared as described [15 (link),29 (link)] in a reaction buffer containing 50 mM Tris-HCl (pH 8.0), 4 mM MgCl₂, 50 mM NaCl, 0.2 mM DTT, 200 mM NAD⁺ (Trevigen) and 130 ng activated DNA (BPS Bioscience). For the PAR hydrolysis activity assays 0.5 μM PARylated PARP1 substrate was used in 10 μL reaction. We estimate that the average number of the ADP-ribose units in chains linked to a PARP1 protein prepared this way is 40 units per protein, giving an approximate concentration of 20 µM monomeric units in the reaction. For the MARylated PARP1, 2 μM of PARP1-E988Q was used as a substrate. Reactions were stopped by the addition of PARP1 inhibitor Olaparib (1 μM). The MgCl₂ (Sigma) concentration was adjusted to 15 mM to allow full Nudix hydrolase activity. Automodified PARP1 was then incubated for 3 hours 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 fractionated by NuPAGE Novex Bis-Tris 4-12% Gel using MOPS buffer (Invitrogen). PARP1-E988Q experiments were visualized by autoradiography, experiments using wild-type PARP1 were visualized by Western blot using a specific anti-PAR antibody.