Resveratrol Inhibits Nucleosome Binding and Catalytic Activity of PARP1

Abstract

The natural polyphenol resveratrol is a biologically active compound that interacts with DNA and affects the activity of some nuclear enzymes. Its effect on the interaction between nucleosomes and poly(ADP-ribose) polymerase-1 (PARP1) and on the catalytic activity of PARP1 was studied using Western blotting, spectrophotometry, electrophoretic mobility shift assay, and single particle Förster resonance energy transfer microscopy. Resveratrol inhibited PARP1 activity at micro- and sub-micromolar concentrations, but the inhibitory effect decreased at higher concentrations due to the aggregation of the polyphenol. The inhibition of PARP1 by resveratrol was accompanied by its binding to the enzyme catalytic center and a subsequent decrease in PARP1 affinity to nucleosomal DNA. Concurrent binding of talazoparib to the substrate binding pocket of PARP1, which occurs in the presence of resveratrol, restores the interaction of PARP1 with nucleosomes, suggesting that the binding sites of resveratrol and talazoparib overlap. The data suggest that resveratrol can be classified as a natural inhibitor of PARP1.

Keywords: PARP1; Western blotting; absorption spectroscopy; gel-shift analysis; nucleosome; poly(ADP-ribose) polymerase-1; resveratrol; spFRET.

Figure 1

Effect of RSV on the interaction between PARP1 and nucleosomes and on PARP1 activity. (A) The experimental approach. Small pink and green circles show the positions of fluorescent labels Cy3 and Cy5 on the nucleosomal DNA. Different domains of PARP1 are shown in color to highlight the multidomain structure of the enzyme. (B) Western blotting analysis of the effect of RSV on the PARP1 activity. Antibodies to poly(ADP-ribose) chains were used to stain auto-PARylated PARP1 after nucleosome-activated reaction of PARylation in the mixture of nucleosomes (Nuc, 2 nM), PARP1 (40 nM), NAD+ (20 μM) and different concentrations of RSV. M: protein molecular weight markers. Similar data were obtained in three independent experiments. (C) Absorption spectra of RSV at different concentrations in an aqueous solution containing 25% dimethyl sulfoxide. The dashed line shows the RSV spectrum at 100 nM magnified ×10 times in intensity.

Figure 2

RSV reduces the binding of PARP1 to nucleosomes. (A) E_PR profiles of nucleosomes (Nuc) and their complexes with PARP1 (40 nM) in the absence and presence of RSV (1, 10 and 100 μM). E_PR profiles are presented as mean ± standard error of the mean (n = 3). (B) EMSA data for nucleosomes and nucleosome–PARP1 complexes in the absence and presence of RSV (1, 10, and 100 μM).

Figure 3

Effect of RSV and TAL on the interaction between PARP1 and nucleosomes. E_PR profiles of nucleosomes (Nuc) and their complexes with PARP1 (40 nM) in the absence and presence of RSV (10 μM) and TAL (1 μM). E_PR profiles are presented as mean ± standard error of the mean (n = 3). (A) PARP1 was pre-incubated with RSV and then incubated with nucleosomes and TAL. (B) PARP1 was pre-incubated with TAL and then incubated with nucleosomes and RSV.