title: A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.
abstract: 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 G2 + 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 more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.
authors.affiliation: Ecole Supérieure de Biotechnologie de Strasbourg, UPR 9003 du Centre National de la Recherche Scientifique, Illkirch-Graffenstaden, France.
authors.forename: V
authors.initials: V
authors.lastname: Schreiber
authors.forename: D
authors.initials: D
authors.lastname: Hunting
authors.forename: C
authors.initials: C
authors.lastname: Trucco
authors.forename: B
authors.initials: B
authors.lastname: Gowans
authors.forename: D
authors.initials: D
authors.lastname: Grunwald
authors.forename: G
authors.initials: G
authors.lastname: De Murcia
authors.forename: J M
authors.initials: JM
authors.lastname: De Murcia
mesh_terms: D017209:Apoptosis; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression; D005799:Genes, Dominant; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D016296:Mutagenesis; D011065:Poly(ADP-ribose) Polymerases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D012854:Sister Chromatid Exchange; D013997:Time Factors
chemical_list: D004268:DNA-Binding Proteins; D008769:Methylnitronitrosoguanidine; D011065:Poly(ADP-ribose) Polymerases