Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells

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-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents. To determine its biological function, we have inactivated both alleles by gene targeting in mice. Treatment of PARP-/- mice either by the alkylating agent N-methyl-N-nitrosourea (MNU) or by gamma-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body gamma-irradiation (8 Gy) mutant mice died rapidly from acute radiation toxicity to the small intestine. Mice-derived PARP-/- cells displayed a high sensitivity to MNU exposure: a G2/M arrest in mouse embryonic fibroblasts and a rapid apoptotic response and a p53 accumulation were observed in splenocytes. Altogether these results demonstrate that PARP is a survival factor playing an essential and positive role during DNA damage recovery.

Figure 1

Inactivation of PARP by homologous recombination. (A) Scheme of targeting construct (Top), the PARP gene and hybridation probe (Middle), and the targeted allele (Bottom). EcoRI restriction was used to detect the targeted gene as indicated. (B) Southern blot of EcoRI-digested tail DNA from wild-type (wt) (+/+), heterozygous (+/−), and homozygous (−/−) PARP-targeted mice, using the 5′probe. The wt and mutant fragment are 9.6 and 3.3 kb, respectively. (C and D) PARP protein was not expressed and poly(ADP-ribose) activity was not detectable in PARP^−/− cells isolated from spleen. B, BamHI; E, EcoRI; X, XhoI; Xb, XbaI; pGK-neo, neomycin-resistance gene driven by the pGK promoter; HSV-Tk, thymidine kinase gene driven by the herpes simplex virus promoter; PARP^+/−, heterozygous PARP mutant; PARP^−/−, homozygous PARP mutant.

Figure 2

Survival of PARP^+/+ and PARP^−/− mice after i.p. injection of MNU at 75 mg/kg body weight at 6 weeks of age (A), and γ-radiation with 8 Gy at 6–8 weeks of age (B). The percentage of alive mice at the end of a week is plotted against age.

Figure 3

Transverse histological sections through the duodenum of an irradiated PARP^−/− mouse (A and D), of an irradiated PARP^+/+ mouse (B and E), and of an untreated PARP^−/− mouse (C and F). (A–C) Full thickness of the duodenal wall. (D–F) Details of the epithelium near the tips of the villi. Note that the untreated PARP^−/− duodenum (C and F) is histologically indistinguishable from its wt counterpart. a, Absorptive cell; c, crypt; g, goblet cell; l, lumen of the small intestine; m, muscularis; v, villi. (A–C, ×170; and D–F, ×750.)

Figure 4

Mean number of SCEs per cell in PARP^−/− and PARP^+/+ mice, before and after exposure to MNU during 9 or 30 h, and mean number of chromatid breaks after exposure to γ-rays 3 or 7 h before harvesting bone marrow cells.

Figure 5

PARP deficiency affects cell cycle progression and activates the programmed cell death following MNU treatment. (A) Cell cycle progression of primary fibroblasts PARP^+/+ and PARP^−/− following mock or MNU treatment. (B) Time course induction of apoptosis in splenocytes lacking PARP by 2 mM MNU. (C) p53 accumulation.