PARP1-DNA Binding Assay by DSB-FA

The effects of the tested compounds on PARP1‐DNA binding were determined by the DSB fluorescence polarization (FA) model as described previously (Chen et al, 2019a (link)). Briefly, the binding reactions were carried out in a buffer containing 12 mM HEPES (pH 8.0), 60 mM KCl, 0.12 mM EDTA, 5.5 μM β‐mercaptoethanol, 8 mM MgCl₂, 0.05 mg/ml BSA and 4% glycerol. Reactions contained a 10 nM DNA probe and 250 nM purified His‐PARP1 protein with DMSO or thioparib. The sequence of the DNA strand used in the assay was 5′‐GGGTTGCGGCCGCTTGGG‐3′ that carried 6‐carboxyfluorescein on the 5′‐terminus, and it was annealed to its complementary DNA strand. The reaction was started with 1 mM NAD⁺ and the data were collected at 60 min with a Synergy H1 microplate reader (BioTek, Winooski, VT).