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; NAD(+):poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents, To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans dominant inhibition of resident PARP activity, As a control, a cell line 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 cells 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 doubling time, a G(2) + M accumulation, and a marked reduction in cell survival, However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD, These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges, Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves mote;than its DNA-binding function, Altogether, these results strongly suggest that PARP is an element of the G(2) checkpoint in mammalian cells.