The addition to proteins of the negatively charged polymer of ADP-ribose (PAR), which is synthesized by PAR polymerases (PARPs) from NAD(+), is a unique post-translational modification. It regulates not only cell survival and cell-death programmes, but also an increasing number of other biological functions with which novel members of the PARP family have been associated. These functions include transcriptional regulation, telomere cohesion and mitotic spindle formation during cell division, intracellular trafficking and energy metabolism.

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.

SummaryPoly(ADP-ribosyl)ation is an immediate DNA-damagedependent post-translational modification of histones and other nuclear proteins that contributes to the survival of injured proliferating cells. Poly(ADP-ribose) polymerases (PARPs) now constitute a large family of 18 proteins, encoded by different genes and displaying a conserved catalytic domain in which PARP-1 (113 kDa), the founding member, and PARP-2 (62 kDa) are so far the sole enzymes whose catalytic activity has been shown to be immediately stimulated by DNA strand breaks. A large repertoire of sequences encoding novel PARPs now extends considerably the field of poly(ADP-ribosyl)ation reactions to various aspects of the cell biology including cell proliferation and cell death. Some of these new members interact with each other, share common partners and common subcellular localizations suggesting possible fine tuning in the regulation of this posttranslational modification of proteins. This review summarizes our present knowledge of this emerging superfamily, which might ultimately improve pharmacological strategies to enhance both antitumor efficacy and the treatment of a number of inflammatory and neurodegenerative disorders. A provisional nomenclature is proposed.

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a zinc-finger DNA-binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents. In response to these breaks, the immediate poly(ADP-ribosyl)ation of nuclear proteins involved in chromatin architecture and DNA metabolism converts DNA damage into intracellular signals that can activate DNA repair programs or cell death options. To have greater insight into the physiological function of this enzyme, we have used the two-hybrid system to find genes encoding proteins putatively interacting with PARP. We have identified a physical association between PARP and the base excision repair (BER) protein XRCC1 (X-ray repair cross-complementing 1) in the Saccharomyces cerevisiae system, which was further confirmed to exist in mammalian cells. XRCC1 interacts with PARP by its central region (amino acids 301 to 402), which contains a BRCT (BRCA1 C terminus) module, a widespread motif in DNA repair and DNA damage-responsive cell cycle checkpoint proteins. Overexpression of XRCC1 in Cos-7 or HeLa cells dramatically decreases PARP activity in vivo, reinforcing the potential protective function of PARP at DNA breaks. Given that XRCC1 is also associated with DNA ligase III via a second BRCT module and with DNA polymerase ␤, our results provide strong evidence that PARP is a member of a BER multiprotein complex involved in the detection of DNA interruptions and possibly in the recruitment of XRCC1 and its partners for efficient processing of these breaks in a coordinated manner. The modular organizations of these interactors, associated with small conserved domains, may contribute to increasing the efficiency of the overall pathway.The genomic integrity of cells is controlled by a network of protein factors that assess the status of the genome and either cause progression of proliferation or induce a halt in the cell cycle. In eukaryotes, DNA strand breaks, introduced either directly by ionizing radiation or indirectly following enzymatic incision of a DNA lesion, trigger the synthesis of poly(ADPribose) by the enzyme poly(ADP-ribose) polymerase (PARP) (1,13,39). At the site of breakage, PARP catalyzes the transfer of the ADP-ribose moiety from its substrate, NAD ϩ , to a limited number of protein acceptors involved in chromatin architecture and DNA metabolism, including the enzyme itself. These modified proteins, which carry long chains of negatively charged ADP-ribose polymers, lose their affinity for DNA and are thus inactivated. The short half-life of the polymer is attributed to the high activity of poly(ADP-ribose) glycohydrolase, which cleaves the ribose-ribose bond (28, 30). Therefore, poly(ADP-ribosylation) is an immediate but transient postranslational modification of nuclear proteins, induced by DNA-damaging agents.The physiological role of PARP has been much debated in the last decade, but recent molecular and genetic approaches, including expression of either a dominant-negative mutant (26,36,44) or antisense oligonucleo...