Changes in poly(ADP-ribose) level modulate the kinetics of DNA strand break rejoining

Abstract

ADP-ribose polymers are rapidly synthesized in cell nuclei by the poly(ADP-ribose) polymerases PARP-1 and PARP-2 in response to DNA strand interruptions, using NAD + as precursor. The level of induced poly(ADP-ribose) formation is proportional to the level of DNA damage and can be decreased by NAD + or PARP deficiency, followed by poor DNA repair and genomic instability. Here we studied the correlation between poly(ADP-ribose) level and DNA strand break repair in lymphoblastoid Raji cells. Poly(ADP-ribose) synthesis was induced by 100 μM H 2O 2 and intensified by the 1,4-dihydropyridine derivative AV-153. The level of poly(ADP-ribose) in individual cells was analyzed by quantitative in situ immunofluorescence and confirmed in whole-cell extracts by Western blotting, and DNA damage was assessed by alkaline comet assays. Cells showed a ∼100-fold increase in poly(ADP-ribose) formation during the first 5 min of recovery from H 2O 2 treatment, followed by a gradual decrease up to 15 min. This synthesis was completely inhibited by the PARP inhibitor NU1025 (100 μM) while the cells treated with AV-153, at non-genotoxic concentrations of 1 nM–10 μM, showed a concentration-dependent increase of poly(ADP-ribose) level up to 130% after the first minute of recovery. The transient increase in poly(ADP-ribose) level was strongly correlated with the speed and efficiency of DNA strand break rejoining (correlation coefficient r ≥ 0.92, p < 0.05). These results are consistent with the idea that poly(ADP-ribose) formation immediately after genome damage reflects rapid assembly and efficient functioning of repair machinery.