TRANSDOMINANT INHIBITION OF POLY(ADP-RIBOSYL)ATION SENSITIZES CELLS AGAINST GAMMA-IRRADIATION AND N-METHYL-N'-NITRO-N-NITROSOGUANIDINE BUT DOES NOT LIMIT DNA-REPLICATION OF A POLYOMAVIRUS REPLICON

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30), with NAD(+) serving as the substrate. PARP is strongly activated upon recognition of DNA strand breaks by its DNA-binding domain. Experimen ts with low-molecular-weight inhibitors of PARP have led to the view t hat PARP activity plays a role in DNA repair and possibly also in DNA replication, cell proliferation, and differentiation. Accumulating evi dence for nonspecific inhibitor effects prompted us to develop a molec ular genetic system to inhibit PARP in living cells, i.e., to overexpr ess selectively the DNA-binding domain of PARP as a dominant negative mutant. Here we report on a cell culture system which allows inducible , high-level expression of the DNA-binding domain. Induction of this d omain leads to about 90% reduction of poly(ADP-ribose) accumulation af ter gamma-irradiation and sensitizes cells to the cytotoxic effect of gamma-irradiation and of N-methyl-N'-nitro-N-nitrosoguanidine. In cont rast, induction does not affect normal cellular proliferation or the r eplication of a transfected polyomavirus replicon, Thus, trans-dominan t inhibition of the poly(ADP-ribose) accumulation occurring after gamm a-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine is specifically associated with a disturbance of the cellular recovery from the inflic ted damage.