Changes in poly(ADP-ribose) level modulate the kinetics of DNA strand break rejoining.
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 microM H(2)O(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 approximately 100-fold increase in poly(ADP-ribose) formation during the first 5 min of recovery from H(2)O(2) treatment, followed by a gradual decrease up to 15 min. This synthesis was completely inhibited by the PARP inhibitor NU1025 (100 microM) while the cells treated with AV-153, at non-genotoxic concentrations of 1 nM-10 microM, 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 > or = 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.
Ryabokon
Nadezhda I
NI
Department of Experimental and Clinical Radiobiology, M Sklodowska-Curie Memorial Cancer Center and Institute, Wybrzeze Armii Krajowej 15, 44-101, Gliwice, Poland.
Goncharova
Rose I
RI
Duburs
Gunars
G
Hancock
Ronald
R
Rzeszowska-Wolny
Joanna
J
D000246:Adenosine Diphosphate Ribose / Q000096:biosynthesis / Q000378:metabolism*; D002460:Cell Line; D053960:DNA Breaks; D004249:DNA Damage; D004260:DNA Repair; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D013997:Time Factors
D000246:Adenosine Diphosphate Ribose; D006861:Hydrogen Peroxide