trans-Dominant Inhibition of Poly(ADP-Ribosyl)ation Sensitizes Cells against γ-Irradiation and N-Methyl-N′-Nitro-N-Nitrosoguanidine but Does Not Limit DNA Replication of a Polyomavirus Replicon

Abstract

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. Experiments with low-molecular-weight inhibitors of PARP have led to the view that PARP activity plays a role in DNA repair and possibly also in DNA replication, cell proliferation, and differentiation. Accumulating evidence for nonspecific inhibitor effects prompted us to develop a molecular genetic system to inhibit PARP in living cells, i.e., to overexpress 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 domain leads to about 90% reduction of poly(ADP-ribose) accumulation after gamma-irradiation and sensitizes cells to the cytotoxic effect of gamma-irradiation and of N-methyl-N'-nitro-N-nitrosoguanidine. In contrast, induction does not affect normal cellular proliferation or the replication of a transfected polyomavirus replicon. Thus, trans-dominant inhibition of the poly(ADP-ribose) accumulation occurring after gamma-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine is specifically associated with a disturbance of the cellular recovery from the inflicted damage.