Hparp 1

Cell-free human PARP-1 enzyme activity was determined in a coupled enzyme assay with fluorometric read-out. In a first step, 200 nM NAD⁺ were mixed with the PARP mix consisting of 1.25 μg activated (partly cleaved) DNA (Sigma#D4522) and 1.0 U/well hPARP-1 (Trevigen #4668-100-01) in assay buffer (50 mM Tris and 2 mM MgCl₂ (pH 8.0)). Subsequently, MP1032 or 4-ANI prepared in assay buffer were added to the reaction mixture. Each assay was performed with a NAD⁺ standard curve (0–100 nM NAD⁺) and a PARP minus control for data analysis. After 90 min, an alcohol dehydrogenase/diaphorase mix consisting of 50 mU alcohol dehydrogenase, 5 mU diaphorase, 50 μM resazurin, and 2% EtOH (v/v) was added. Fluorescence kinetics (5 min steps for 40 min) was measured using a Synergy2 plate reader (BioTek, Bad Friedrichshall, Germany) with λ_exc 540/25 nm, λ_em 590/20 nm, and a 550 nm mirror. Data were calculated using NAD⁺ standard curve and expressed as percent hPARP-1 inhibition.

For the analysis of TRF1 PARylation by PARP-1^{31 (link)}, 160 ng of wt or delta acidic hTRF1/sample were incubated in the reaction buffer containing 5 units of hPARP-1 (High Specific Activity, Trevigen), 2.5 μg DNase I-activated calf thymus DNA, 200 mM NAD+, Tris-HCl pH 8, 10 mM MgCl₂ and 2 mM dithiothreitol. After 30 min of incubation at 25 °C, the reaction was stopped by the addition of a Laemmli sample buffer and samples were analyzed by gel electrophoresis on 8% SDS-PAGE and Western blot. PARylated PARP1, hTRF1 or delta acidic hTRF1 were detected using anti-PAR monoclonal antibody (Trevigen) and input TRF1 was revealed with anti-His (anti 6-His Rabbit Pab Sigma Aldrich) or anti TRF1 antibody (rabbit Pab sc-6165, Santa Cruz). For the detection of biotin-labeled PARylated proteins the same assay was conducted in presence of biotin-NAD + (Sigma Aldrich) followed by SDS-PAGE and western blot detection with anti-streptavidin HRP antibody (Molecular Probes).