A DOMINANT-NEGATIVE MUTANT OF HUMAN POLY(ADP-RIBOSE) POLYMERASE AFFECTS CELL RECOVERY, APOPTOSIS, AND SISTER-CHROMATID EXCHANGE FOLLOWING DNA-DAMAGE

Poly(ADP-ribose) polymerase [PARP; NAD(+) ADP-ribosyltransferase; (+): poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferas e, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein tha t specifically recognizes DNA strand breaks produced by various genoto xic agents, To study the biological function of this enzyme, we have e stablished stable HeLa cell lines that constitutively produce the 46-k Da DNA-binding domain of human PARP (PARP-DBD), leading to the trans d ominant inhibition of resident PARP activity, As a control, a cell lin e was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro, Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for c ells treated with genotoxic agents, Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl -N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their d oubling time, a G(2) + M accumulation, and a marked reduction in cell survival, However, UVC irradiation had no preferential effect on the c ell growth or viability of cell lines expressing the PARP-DBD, These P ARP-DBD-expressing cells treated with MNNG presented the characteristi c nucleosomal DNA ladder, one of the hallmarks of cell death by apopto sis. Moreover, these cells exhibited chromosomal instability as demons trated by higher frequencies of both spontaneous and MNNG-induced sist er chromatid exchanges, Surprisingly, the line producing the mutated D BD had the same behavior as those producing the wt DBD, indicating tha t the mechanism of action of the dominant-negative mutant involves mot e;than its DNA-binding function, Altogether, these results strongly su ggest that PARP is an element of the G(2) checkpoint in mammalian cell s.