Overexpression of the DNA-binding domain of poly(ADP-ribose) polymerase inhibits rejoining of ionizing radiation-induced DNA double-strand breaks

Abstract

Purpose : To assess the influence of trans -dominant inhibition of poly(ADP-ribosyl)ation on the rejoining kinetics of radiation-induced DNA double-strand breaks (DSB). Materials and methods : Stable transfectants of the SV40-transformed hamster cell line CO60 were used: COM3 cells contain a construct to overexpress the poly(ADP-ribose) polymerase (PARP-1) DNA-binding domain (DBD) when induced by dexamethasone, as well as a construct for the constitutive overexpression of the human glucocorticoid receptor (Hg0). COR3 are control cells containing only the Hg0 plasmid. DSB induction and rejoining in X-irradiated cells was assessed by DNA pulsed-field electrophoresis. Results : DSB induction was identical in both cell lines and independent of the presence of dexamethasone. DSB rejoining kinetics was independent of dexamethasone in COR3 cells and identical to COM3 cells without dexamethasone. However, in COM3 cells treated with dexamethasone to induce PARP-1 DBD overexpression, the fast component of the rejoining kinetic was largely reduced, and residual fragmentation increased concomitant with the increased damage fraction in slow rejoining. Conclusions : The results indicate that inhibition of cellular PARP-1 does not affect the rate-limiting step of either fast or slow DSB rejoining. Rather, it appears that absence of poly(ADP-ribosyl)ation due to dominant negative PARP-1 expression induces a shift from rapid to slow DSB rejoining and by this mechanism PARP inhibition may increase the risk of repair failures.