or by ␥-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body ␥-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 G 2 ͞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.To protect their genome from the deleterious consequences of accumulation of unrepaired or misrepaired lesions, cells have developed an intricate DNA damage surveillance network. Through its function as a single-stranded breaks detector, poly(ADP-ribose) polymerase [PARP; NAD ϩ ADP-ribosyltransferase; NAD ϩ : poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30], a nuclear enzyme, participates to this basic process (1). PARP (113 kDa) has a modular organization (2): a N-terminal DNA-binding domain that acts as a molecular nick-sensor, encompassing two zinc-finger motifs (3) and a bipartite nuclear location signal (4), a central region bearing the auto-poly(ADP-ribosylation) sites which serves to regulate PARP-DNA interactions and a C-terminal catalytic domain involved in the nick-binding dependent poly(ADP-ribose) synthesis (5). The x-ray crystallographic structure of this domain has been recently solved revealing a surprising structural homology between the active site of PARP and that of bacterial mono-ADP-ribosylating toxins despite weak sequence homology (6).Although the physiological role of PARP is still much debated, recent molecular and genetic approaches including expression of either a dominant-negative mutant (7-10) or antisense (11) have clearly revealed the implication of PARP in the maintenance of the genomic integrity in the base excision repair pathway (7)(8)(9)(10)12). To elucidate its function we disrupted the mouse PARP gene by homologous recombination and exposed the PARP-deficient mice and derived cells to various genotoxins. MATERIALS AND METHODSGene Targeting in Embryonic Stem Cells and Generation of Mice. Mouse PARP was isolated from a 129SVJ strain genomic library. The targeting vector was constructed using a 9-kb EcoRI fragment extending from intron 2 to 7 by inserting PGK-neo (phosphoglycerate kinase promoter followed by the neo gene) in the BamHI site of the 4th exon and herpes simplex virus thymidine kinase followed by the TK gene (HSV-Tk) in the XhoI site outside the sequence of the targeting vector. Following electroporation, embryonic stem cells were selected in 200 g⅐ml Ϫ1 G418 and 2 mM of gancyclovir. A positive clone microinjected into C57BL͞6 blastocysts (13) gave rise to chimaeric offspring, which in turn were mated with C57BL͞6.

The DNA damage-dependent poly(ADP-ribose) polymerases, PARP-1 and PARP-2, homo-and heterodimerize and are both involved in the base excision repair (BER) pathway. Here, we report that mice carrying a targeted disruption of the PARP-2 gene are sensitive to ionizing radiation. Following alkylating agent treatment, parp-2 ±/± -derived mouse embryonic ®broblasts exhibit increased post-replicative genomic instability, G 2 /M accumulation and chromosome mis-segregation accompanying kinetochore defects. Moreover, parp-1 ±/± parp-2 ±/± double mutant mice are not viable and die at the onset of gastrulation, demonstrating that the expression of both PARP-1 and PARP-2 and/or DNA-dependent poly(ADP-ribosyl) ation is essential during early embryogenesis. Interestingly, speci®c female embryonic lethality is observed in parp-1 +/± parp-2 ±/± mutants at E9.5. Metaphase analyses of E8.5 embryonic ®broblasts highlight a speci®c instability of the X chromosome in those females, but not in males. Together, these results support the notion that PARP-1 and PARP-2 possess both overlapping and non-redundant functions in the maintenance of genomic stability.

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The crystal structures of the catalytic frag-

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