Thermal Shift Assay for PARP-Family Enzymes

DSF experiments were performed as described^{8 (link), 13 (link)} using 5 µM protein, 2.5 µM DNA when indicated and various concentrations of compound (BAD, carba-NAD⁺, benzamide, and ADP-ribose) as indicated in Fig. 1 and Fig. 3. Experiments were performed on a Roche LightCycler 480 RT-PCR. Reactions including DNA were performed with a 26- or 28-base pair (bp) unphosphorylated oligonucleotide for PARP-1, a 28-bp 5´-phosphorylated (5´P) oligonucleotide for PARP-2, and a 47-bp oligonucleotide including a central 5´P nick for PARP-3¹³ in the following buffer: 25 mM Hepes pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.1 mM TCEP. Reactions with PARP-3 were conducted at lower ionic strength to allow binding to DNA as described (25 mM Hepes pH 8.0, 50 mM NaCl, 1 mM EDTA and 0.1 mM TCEP)^{13 (link)}. ΔT_M values were calculated by subtracting the T_M determined for the protein in the absence of compound from the T_M determined in the presence of compound. Experiments were performed in triplicate and a Boltzmann sigmoid was fit to the data to determine the T_M values (KaleidaGraph).