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The peroxisome proliferator-activated receptor-gamma (PPARgamma, NR1C3) in complex with the retinoid X receptor (RXR) plays a central role in white adipose tissue (WAT) differentiation and function, regulating the expression of key WAT proteins. In this report we show that poly(ADP-ribose) polymerase-2 (PARP-2), also known as an enzyme participating in the surveillance of the genome integrity, is a member of the PPARgamma/RXR transcription machinery. PARP-2(-/-) mice accumulate less WAT, characterized by smaller adipocytes. In the WAT of PARP-2(-/-) mice the expression of a number of PPARgamma target genes is reduced despite the fact that PPARgamma1 and -gamma2 are expressed at normal levels. Consistent with this, PARP-2(-/-) mouse embryonic fibroblasts fail to differentiate to adipocytes. In transient transfection assays, PARP-2 small interference RNA decreases basal activity and ligand-dependent activation of PPARgamma, whereas PARP-2 overexpression enhances the basal activity of PPARgamma, although it does not change the maximal ligand-dependent activation. In addition, we show a DNA-dependent interaction of PARP-2 and PPARgamma/RXR heterodimer by chromatin immunoprecipitation. In combination, our results suggest that PARP-2 is a novel cofactor of PPARgamma activity.

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a critical role in endothelial cell dysfunction associated with various pathophysiological conditions. To elucidate PARP-1 pathways involved in endothelial cell dysfunction, it is essential to establish "in vitro" experimental models using isolated endothelial cells. So far, two approaches have been used: primary endothelial cells from PARP-1-/- mice which have a limited life-span, being a major handicap if large quantities of cells are required; and pharmacological inhibition of PARP in PARP-1+/+ endothelial cell lines, which is not specific for PARP-1 and would have biological effects different that genetic inhibition. To overcome these limitations, we have established an immortalized PARP-1-/- endothelial cell line (HYKO6) by transfection of primary cells with a plasmid containing the SV40 genome and selected on the basis of morphological and phenotypical features. The HYKO6 cell line exhibited endothelial characteristics, such as constitutive expression of CD105, CD31, ICAM-2, VCAM-1, and von Willebrand factor and formation of capillary-like structures (CLS) on Matrigel surface. However, expression of ICAM-1 antigen is lost in the HYKO6 cells. After TNF-alpha treatment, HYKO6 cells exhibited increased expression of E-selectin and VCAM-1. Likewise, NF-kappaB-dependent transcriptional activation was increased in the HYKO6 cell line in response to TNF-alpha at a level similar to that found for primary PARP-1-/- cells. This cell line should provide, for the first time, a valuable tool to study PARP-1 pathways in endothelial cell dysfunction.

Caspase-8 is the prototypic initiator of the death domain receptor pathway of apoptosis. Here, we report that caspase-8 not only triggers and amplifies the apoptotic process at cytoplasmic sites but can also act as an executioner at nuclear levels. In a murine model of acute ischemia, caspase-8 is relocated into the nucleus of apoptotic neurons, where it cleaves PARP-2, a member of the poly(ADP-ribose) polymerase family involved in DNA repair. As indicated by site-directed mutagenesis, PARP-2 cleavage occurs preferentially at the LQMD sequence mapped between the DNA binding and the catalytic domains of the protein. This is close to the cleavage sequence found in Bid, the cytoplasmic target of caspase-8. Activity assays confirm that cleavage of PARP-2 results in inactivation of its poly(ADP-ribosylation) property, proportional to the efficiency of the cleavage. Our findings add to the complexity of proteolytic caspase networks by demonstrating that caspase-8 is in turn an initiator, amplifier, and effector caspase.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that consumes NAD in response to DNA strand breaks. Its excessive activation seems particularly deleterious to pancreatic β-cells, as exemplified by the complete resistance of PARP-1-deficient mice to the toxic diabetes induced by streptozotocin. Because of the possible implication of this enzyme in type 1 diabetes, many human trials using nicotinamide, an inhibitor of PARP-1, have been conducted either in patients recently diagnosed or in subjects highly predisposed to this disease. To analyze the role of this enzyme in murine type 1 diabetes, we introgressed a disrupted PARP-1 allele onto the autoimmune diabetes-prone nonobese diabetic (NOD) mouse strain. We showed that these mice were protected neither from spontaneous nor from cyclophosphamide-accelerated diabetes. Surprisingly they were also highly sensitive to the diabetes induced by a single high dose of streptozotocin, standing in sharp contrast with C57BL/6 mice that bear the same inactivated PARP-1 allele. Our results suggest that NOD mice are characterized not only by their immune dysfunction but also by a peculiarity of their islets leading to a PARP-1-independent mechanism of streptozotocin-induced β-cell death.

Mammalian DNA ligases are composed of a conserved catalytic domain flanked by unrelated sequences. At the C-terminal end of the catalytic domain, there is a 16-amino acid sequence, known as the conserved peptide, whose role in the ligation reaction is unknown. Here we show that conserved positively charged residues at the C-terminal end of this motif are required for enzyme-AMP formation. These residues probably interact with the triphosphate tail of ATP, positioning it for nucleophilic attack by the active site lysine. Amino acid residues within the sequence RFPR, which is invariant in the conserved peptide of mammalian DNA ligases, play critical roles in the subsequent nucleotidyl transfer reaction that produces the DNA-adenylate intermediate. DNA binding by the N-terminal zinc finger of DNA ligase III, which is homologous with the two zinc fingers of poly(ADP-ribose) polymerase, is not required for DNA ligase activity in vitro or in vivo. However, this zinc finger enables DNA ligase III to interact with and ligate nicked DNA at physiological salt concentrations. We suggest that in vivo the DNA ligase III zinc finger may displace poly(ADP-ribose) polymerase from DNA strand breaks, allowing repair to occur.

Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins in response to DNA damage that activates the base excision repair machinery. Poly(ADP-ribose) polymerase which we will now call PARP-1, has been the only known enzyme of this type for over 30 years. Here, we describe a cDNA encoding a 62-kDa protein that shares considerable homology with the catalytic domain of PARP-1 and also contains a basic DNA-binding domain. We propose to call this enzyme poly(ADP-ribose) polymerase 2 (PARP-2). The PARP-2 gene maps to chromosome 14C1 and 14q11.2 in mouse and human, respectively. Purified recombinant mouse PARP-2 is a damaged DNA-binding protein in vitro and catalyzes the formation of poly(ADP-ribose) polymers in a DNA-dependent manner. PARP-2 displays automodification properties similar to PARP-1. The protein is localized in the nucleus in vivo and may account for the residual poly(ADP-ribose) synthesis observed in PARP-1-deficient cells, treated with alkylating agents or hydrogen peroxide.

To ensure the accurate transmission of genetic information in dividing cells, specific biochemical pathways maintain integrity. Fundamental to these pathways is the recognition, by specific proteins, of genomic lesions, which signal the presence of DNA damage to other nuclear and cytoplasmic factors. DNA strand breaks, generated either directly by genotoxic agents (oxygen radicals, ionizing radiations, or monofunctional alkylating agents) or indirectly after enzymatic incision of a DNA-base lesion, trigger the synthesis of poly(ADP-ribose) by the enzyme poly(ADP-ribose) polymerase (PARP [E.C.2.4.2.30]).

Poly(ADP-ribose) polymerase (PARP) is a constitutive factor of the DNA damage surveillance network in dividing cells. Based on its capacity to bind to DNA strand breaks, PARP plays a regulatory role in their resolution in vivo. ATM belongs to a large family of proteins involved in cell cycle progression and checkpoints in response to DNA damage. Both proteins may act as sensors of DNA damage to induce multiple signalling pathways leading to activation of cell cycle checkpoints and DNA repair. To determine a possible relationship between PARP and ATM, we examined the PARP response in an ATM-null background. We demonstrated that ATM deficiency does not affect PARP activity in human cell lines or Atm-deficient mouse tissues, nor does it alter PARP activity induced by oxidative damage or gamma-irradiation. Our results support a model in which PARP and ATM could be involved in distinct pathways, both effectors transducing the damage signal to cell cycle regulators.

We have studied the apoptotic response of poly(ADP-ribose) polymerase (PARP)-/- cells to different inducers and the consequences of the expression of an uncleavable mutant of PARP on the apoptotic process. The absence of PARP drastically increases the sensitivity of primary bone marrow PARP-/- cells to apoptosis induced by an alkylating agent but not by a topoisomerase I inhibitor CPT-11 or by interleukin-3 removal. cDNA of wild type or of an uncleavable PARP mutant (D214A-PARP) has been introduced into PARP-/- fibroblasts, which were exposed to anti-CD95 or an alkylating agent to induce apoptosis. The expression of D214A-PARP results in a significant delay of cell death upon CD95 stimulation. Morphological analysis shows a retarded cell shrinkage and nuclear condensation. Upon treatment with an alkylating agent, expression of wild-type PARP cDNA into PARP-deficient mouse embryonic fibroblasts results in the restoration of the cell viability, and the D214A-PARP mutant had no further effect on cell recovery. In conclusion, PARP-/- cells are extremely sensitive to apoptosis induced by triggers (like alkylating agents), which activates the base excision repair pathway of DNA, and the cleavage of PARP during apoptosis facilitates cellular disassembly and ensures the completion and irreversibility of the process.

To investigate the physiological function of poly(ADP-ribose) polymerase (PARP), we used a gene targeting strategy to generate mice lacking a functional PARP gene. These PARP -/- mice were exquisitely sensitive to the monofunctional-alkylating agent N -methyl- N -nitrosourea (MNU) and gamma-irradiation. In this report, we have analysed the cause of this increased lethality using primary and/or spontaneously immortalized mouse embryonic fibroblasts (MEFs) derived from PARP -/- mice. We found that the lack of PARP renders cells significantly more sensitive to methylmethanesulfonate (MMS), causing cell growth retardation, G2/M accumulation and chromosome instability. An important delay in DNA strand-break resealing was observed following treatment with MMS. This severe DNA repair defect appears to be the primary cause for the observed cytoxicity of monofunctional-alkylating agents, leading to cell death occurring after G2/M arrest. Cell viability following MMS treatment could be fully restored after transient expression of the PARP gene. Altogether, these results unequivocally demonstrate that PARP is required for efficient base excision repair in vivo and strengthens the role of PARP as a survival factor following genotoxic stress.

Kin17 is a 45 kDa protein encoded by the KIN17 gene located on mouse chromosome 2, band A. The kin17 amino acid sequence predicts two domains, which were shown to be functional: (i) a bipartite nuclear localization signal (NLS) that can drive the protein to the cell nucleus, (ii) a bona fide zinc finger of the C2H2 type. The zinc finger is involved in kin17 binding to double-stranded DNA since a mutant deleted of the zinc finger, kin17 delta 1, showed reduced binding. Single-stranded DNA was bound poorly by kin17. Interestingly, we found that kin17 protein showed preferential binding to curved DNA from either pBR322 or synthetic oligonucleotides. Binding of kin17 to a non-curved DNA segment increased after we had inserted into it a short curved synthetic oligonucleotide. Kin17 delta 2, a mutant deleted of 110 amino acids at the C-terminal end, still exhibited preferential binding to curved DNA and so did kin17 delta 1, suggesting that a domain recognizing curved DNA is located in the protein core.

We have overproduced the full-length human poly(ADP-ribose) polymerase (PARP) in Spodoptera frugiperda (Sf9) cells using a baculovirus expression vector system. Approx. 20 mg of purified protein from 5 x 10(8) Sf9 cells were obtained by a simple three-step purification procedure including 3-aminobenzamide affinity chromatography. The recombinant protein (rePARP), which migrates as a unique 116-kDa band on SDS-polyacrylamide gels, was identified as PARP by Western blotting using either polyclonal or monoclonal antibodies raised against the purified human and calf thymus enzymes. Furthermore, rePARP is a functional protein, as demonstrated by its ability to specifically bind Zn2+ and DNA, and to recognize single-strand breaks in DNA. The purified enzyme has the same affinity for NAD+ and turnover number as the human placental PARP. Thus, rePARP produced in insect cells is biologically active and suitable for functional analysis. The reproducibility of the overproduction and the simplicity of the purification protocol, as well as the yield of the produced protein, should greatly facilitate physicochemical and structural studies.

The peroxisome proliferator-activated receptor-gamma (PPARgamma, NR1C3) in complex with the retinoid X receptor (RXR) plays a central role in white adipose tissue (WAT) differentiation and function, regulating the expression of key WAT proteins. In this report we show that poly(ADP-ribose) polymerase-2 (PARP-2), also known as an enzyme participating in the surveillance of the genome integrity, is a member of the PPARgamma/RXR transcription machinery. PARP-2(-/-) mice accumulate less WAT, characterized by smaller adipocytes. In the WAT of PARP-2(-/-) mice the expression of a number of PPARgamma target genes is reduced despite the fact that PPARgamma1 and -gamma2 are expressed at normal levels. Consistent with this, PARP-2(-/-) mouse embryonic fibroblasts fail to differentiate to adipocytes. In transient transfection assays, PARP-2 small interference RNA decreases basal activity and ligand-dependent activation of PPARgamma, whereas PARP-2 overexpression enhances the basal activity of PPARgamma, although it does not change the maximal ligand-dependent activation. In addition, we show a DNA-dependent interaction of PARP-2 and PPARgamma/RXR heterodimer by chromatin immunoprecipitation. In combination, our results suggest that PARP-2 is a novel cofactor of PPARgamma activity.

Poly(ADP-ribosyl)ation is an immediate DNA damage-dependent posttranslational 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 is immediately stimulated by DNA strand-breaks. This review summarizes our present knowledge of the structure and function of PARP-2, the closest relative to PARP-1.

Changes in chromatin structure emanating from DNA breaks are among the most initiating events in the damage response of the cell. In higher eukaryotes, poly(ADP-ribose) polymerase-1 (PARP-1) translates the occurrence of DNA breaks detected by its zinc-finger domain into a signal, poly ADP-ribose, synthesized and amplified by its DNA-damage dependent catalytic domain. This epigenetic mark on chromatin, induced by DNA discontinuities, is now considered as a part of a survival program aimed at protecting primarily chromatin integrity and stability. In this chapter we describe some of our methods for determining in vivo and in vitro PARP-1 activation in response to DNA strand breaks. Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins induced by DNA strand-breaks that contributes to the survival of injured proliferating cells (D'Amours et al., 1999). 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 is immediately stimulated by DNA strand-breaks (Ame et al., 2004). PARP-1 fulfils several key functions in repairing an interruption of the sugar phosphate backbone. It efficiently detects the presence of a break by its N-terminal zinc-finger domain; the occurrence of a break is immediately translated into a posttranslational modification of histones H1 and H2B leading to chromatin structure relaxation and therefore to increased DNA accessibility. As an amplified DNA damage signal, auto-poly(ADP-ribosyl)ation of PARP-1 triggers the recruitment of XRCC1, which coordinates and stimulates the repair process, to the DNA damage sites in less than 15 s in living cells (Okano et al., 2003). Although dispensable in a test tube DNA repair experiment, in vivo these three properties positively influence the overall kinetics of a DNA damage-detection/signaling pathway leading rapidly to the resolution of DNA breaks. Accordingly, poly ADP-ribose (PAR) synthesis and the accompanying NAD consumption are now considered as bona fide marks of DNA interruptions in the genome. In this chapter we describe several methods for determining PARP activation in response to the occurrence of DNA breaks in vitro and in vivo.

The DNA repair proteins poly(ADP-ribose) polymerase-1 (PARP-1), Ku86, and catalytic subunit of DNA-PK (DNA-PKcs) have been involved in telomere metabolism. To genetically dissect the impact of these activities on telomere function, as well as organismal cancer and aging, we have generated mice doubly deficient for both telomerase and any of the mentioned DNA repair proteins, PARP-1, Ku86, or DNA-PKcs. First, we show that abrogation of PARP-1 in the absence of telomerase does not affect the rate of telomere shortening, telomere capping, or organismal viability compared with single telomerase-deficient controls. Thus, PARP-1 does not have a major role in telomere metabolism, not even in the context of telomerase deficiency. In contrast, mice doubly deficient for telomerase and either Ku86 or DNA-PKcs manifest accelerated loss of organismal viability compared with single telomerase-deficient mice. Interestingly, this loss of organismal viability correlates with proliferative defects and age-related pathologies, but not with increased incidence of cancer. These results support the notion that absence of telomerase and short telomeres in combination with DNA repair deficiencies accelerate the aging process without impacting on tumorigenesis.

Poly(ADP-ribosyl)ation is an immediate DNA damage-dependent posttranslational modification of histones and nuclear proteins that contributes to the survival of injured proliferating cells. Poly(ADP-ribose) polymerases (PARPs) now constitute a superfamily of 18 proteins, encoded by different genes and displaying a common conserved catalytic domain. PARP-1 (113kDa), the founding member, and PARP-2 (62kDa) are both involved in DNA-break sensing and signaling when single strand break repair (SSBR) or base excision repair (BER) pathways are engaged. The generation by homologous recombination of deficient mouse models have confirmed the caretaker function of PARP-1 and PARP-2 in mammalian cells under genotoxic stress. This review summarizes our present knowledge on their physiological role in the cellular response to DNA damage and on the genetic interactions between PARP-1, PARP-2, Atm that play an essential role during early embryogenesis.

Cellular recovery from ionizing radiation (IR)-induced damage involves poly(ADP-ribose) polymerase (PARP-1 and PARP-2) activity, resulting in the induction of a signalling network responsible for the maintenance of genomic integrity. In the present work, a charged particle microbeam delivering 3.2 MeV protons from a Van de Graaff accelerator has been used to locally irradiate mammalian cells. We show the immediate response of PARPs to local irradiation, concomitant with the recruitment of ATM and Rad51 at sites of DNA damage, both proteins being involved in DNA strand break repair. We found a co-localization but no connection between two DNA damage-dependent post-translational modifications, namely poly(ADP-ribosyl)ation of nuclear proteins and phosphorylation of histone H2AX. Both of them, however, should be considered and used as bona fide immediate sensitive markers of IR damage in living cells. This technique thus provides a powerful approach aimed at understanding the interactions between the signals originating from sites of DNA damage and the subsequent activation of DNA strand break repair mechanisms

Ku is an abundant heterodimeric nuclear protein, consisting of 70-kDa and 86-kDa tightly associated subunits that comprise the DNA binding component of DNA-dependent protein kinase. Poly(ADP)ribose polymerase-1 (PARP-1) is a 113-kDa protein that catalyzes the synthesis of poly(ADP-ribose) on target proteins. Both Ku and PARP-1 recognize and bind to DNA ends. Ku functions in the non-homologous end joining (NHEJ) repair pathway whereas PARP-1 functions in the single strand break repair and base excision repair (BER) pathways. Recent studies have revealed that PARP-1 and Ku80 interact in vitro. To determine whether the association of PARP-1 and Ku80 has any physiological significance or synergistic function in vivo, mice lacking both PARP-1 and Ku80 were generated. The resulting offspring died during embryonic development displaying abnormalities around the gastrulation stage. In addition, PARP-1-/-Ku80-/- cultured blastocysts had an increased level of apoptosis. These data suggest that the functions of both Ku80 and PARP-1 are essential for normal embryogenesis and that a loss of genomic integrity leading to cell death through apoptosis is likely the cause of the embryonic lethality observed in these mice.

The DNA damage dependence of poly(ADP-ribose) polymerase-2 (PARP-2) activity is suggestive of its implication in genome surveillance and protection. Here we show that the PARP-2 gene, mainly expressed in actively dividing tissues follows, but to a smaller extent, that of PARP-1 during mouse development. We found that PARP-2 and PARP-1 homo- and heterodimerize; the interacting interfaces, sites of reciprocal modification, have been mapped. PARP-2 was also found to interact with three other proteins involved in the base excision repair pathway: x-ray cross complementing factor 1 (XRCC1), DNA polymerase beta, and DNA ligase III, already known as partners of PARP-1. XRCC1 negatively regulates PARP-2 activity, as it does for PARP-1, while being a polymer acceptor for both PARP-1 and PARP-2. To gain insight into the physiological role of PARP-2 in response to genotoxic stress, we developed by gene disruption mice deficient in PARP-2. Following treatment by the alkylating agent N-nitroso-N-methylurea (MNU), PARP-2-deficient cells displayed an important delay in DNA strand breaks resealing, similar to that observed in PARP-1 deficient cells, thus confirming that PARP-2 is also an active player in base excision repair despite its low capacity to synthesize ADP-ribose polymers.

Poly(ADP-ribose) polymerase (PARP)-1, a detector of single-strand breaks, plays a key role in the cellular response to DNA damage. PARP-1–deficient mice are hypersensitive to genotoxic agents and display genomic instability due to a DNA repair defect in the base excision repair pathway. A previous report suggested that PARP-1–deficient mice also had a severe telomeric dysfunction consisting of telomere shortening and increased end-to-end fusions (d'Adda di Fagagna, F., M.P. Hande, W.-M. Tong, P.M. Lansdorp, Z.-Q. Wang, and S.P. Jackson. 1999. Nat. Genet. 23:76–80). In contrast to that, and using a panoply of techniques, including quantitative telomeric (Q)-FISH, we did not find significant differences in telomere length between wild-type and PARP-1−/− littermate mice or PARP-1−/− primary cells. Similarly, there were no differences in the length of the G-strand overhang. Q-FISH and spectral karyotyping analyses of primary PARP-1−/− cells showed a frequency of 2 end-to-end fusions per 100 metaphases, much lower than that described previously (d'Adda di Fagagna et al., 1999). This low frequency of end-to-end fusions in PARP-1−/− primary cells is accordant with the absence of severe proliferative defects in PARP-1−/− mice. The results presented here indicate that PARP-1 does not play a major role in regulating telomere length or in telomeric end capping, and the chromosomal instability of PARP-1−/− primary cells can be explained by the repair defect associated to PARP-1 deficiency. Finally, no interaction between PARP-1 and the telomerase reverse transcriptase subunit, Tert, was found using the two-hybrid assay.

PARP‐1‐deficient mice display a severe defect in the base excision repair pathway leading to radiosensitivity and genomic instability. They are protected against necrosis induced by massive oxidative stress in various inflammatory processes. Mice lacking p53 are highly predisposed to malignancy resulting from defective cell cycle checkpoints, resistance to DNA damage‐induced apoptosis as well as from upregulation of the iNOS gene resulting in chronic oxidative stress. Here, we report the generation of doubly null mutant mice. We found that tumour‐free survival of parp‐1 −/− p53 −/− mice increased by 50% compared with that of parp‐1 +/+ p53 −/− mice. Tumour formation in nude mice injected with oncogenic parp‐1 −/− p53 −/− fibroblasts was significantly delayed compared with parp‐1 +/+ p53 −/− cells. Upon γ‐irradiation, a partial restoration of S‐phase radiosensitivity was found in parp‐1 −/− p53 −/− primary fibroblasts compared with parp‐1 +/+ p53 −/− cells. In addition, iNOS expression and nitrite release were dramatically reduced in the parp‐1 −/− p53 −/− mice compared with parp‐1 +/+ p53 −/− mice. The abrogation of the oxydated status of p53 −/− cells, due to the absence of parp‐1 , may be the cause of the delay in the onset of tumorigenesis in parp‐1 −/− p53 −/− mice.

PARP-1 and ATM are both involved in the response to DNA strand breaks, resulting in induction of a signaling network responsible for DNA surveillance, cellular recovery, and cell survival. ATM interacts with double-strand break repair pathways and induces signals resulting in the control of the cell cycle-coupled checkpoints. PARP-1 acts as a DNA break sensor in the base excision repair pathway of DNA. Mice with mutations inactivating either protein show radiosensitivity and high radiation-induced chromosomal aberration frequencies. Embryos carrying double mutations of both PARP-1 and Atm genes were generated. These mutant embryos show apoptosis in the embryo but not in extraembryonic tissues and die at embryonic day 8.0, although extraembryonic tissues appear normal for up to 10.5 days of gestation. These results reveal a functional synergy between PARP-1 and ATM during a period of embryogenesis when cell cycle checkpoints are not active and the embryo is particularly sensitive to DNA damage. These results suggest that ATM and PARP-1 have synergistic phenotypes due to the effects of these proteins on signaling DNA damage and/or on distinct pathways of DNA repair.

In mammalian cells, damaged bases in DNA are corrected by the base excision repair pathway which is divided into two distinct pathways depending on the length of the resynthesized patch, replacement of one nucleotide for short-patch repair, and resynthesis of several nucleotides for long-patch repair. The involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in both pathways has been investigated by using PARP-1-deficient cell extracts to repair single abasic sites derived from uracil or 8-oxoguanine located in a double-stranded circular plasmid. For both lesions, PARP-1-deficient cell extracts were about half as efficient as wild-type cells at the polymerization step of the short-patch repair synthesis, but were highly inefficient at the long-patch repair. We provided evidence that PARP-1 constitutively interacts with DNA polymerase beta. Using cell-free extracts from mouse embryonic cells deficient in DNA polymerase beta, we demonstrated that DNA polymerase beta is involved in the repair of uracil-derived AP sites via both the short and the long-patch repair pathways. When both PARP-1 and DNA polymerase beta were absent, the two repair pathways were dramatically affected, indicating that base excision repair was highly inefficient. These results show that PARP-1 is an active player in DNA base excision repair.

Poly (ADP-ribose) polymerase-1 is a nuclear DNA-binding protein that participates in the DNA base excision repair pathway in response to genotoxic stress in mammalian cells. Here we show that PARP-1-deficient cells are defective in NF-kappaB-dependent transcription activation, but not in its nuclear translocation, in response to TNF-alpha. Treating mice with lipopolysaccharide (LPS) resulted in the rapid activation of NF-kappaB in macrophages from PARP-1(+/+) but not from PARP-1(-/-) mice. PARP-1-deficient mice were extremely resistant to LPS-induced endotoxic shock. The molecular basis for this resistance relies on an almost complete abrogation of NF-kappaB-dependent accumulation of TNF-alpha in the serum and a down-regulation of inducible nitric oxide synthase (iNOS), leading to decreased NO synthesis, which is the main source of free radical generation during inflammation. These results demonstrate a functional association in vivo between PARP-1 and NF-kappaB, with consequences for the transcriptional activation of NF-kappaB and a systemic inflammatory process.

Poly(ADP-ribose) polymerase (PARP) 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 lesions, the immediate poly(ADP-ribosylation) of nuclear proteins converts DNA interruptions into intracellular signals that activate DNA repair or cell death programs. To elucidate the biological function of PARP in vivo, the mouse PARP gene was inactivated by homologous recombination to generate mice lacking a functional PARP gene. PARP knockout mice and the derived mouse embryonic fibroblasts (MEFs) were acutely sensitive to monofunctional alkylating agents and gamma-irradiation demonstrating that PARP is involved in recovery from DNA damage that triggers the base excision repair (BER) process. To address the issue of the role of PARP in BER, the ability of PARP-deficient mammalian cell extracts to repair a single abasic site present on a circular duplex plasmid molecule was tested in a standard in vitro repair assay. The results clearly demonstrate, for the first time, the involvement of PARP in the DNA synthesis step of the base excision repair process.

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(ADP-ribose) 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+, 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 oligonucleotides (14), have clearly implicated PARP in the base excision repair (BER) pathway. A more definitive assessment of PARP function was recently provided by the generation of PARP-deficient mice by homologous recombination (35, 53). We found that PARP−/− mice are hypersensitive to monofunctional alkylating agents and γ-irradiation and display a marked genomic instability (sister chromatid exchanges and chromatid and chromosome breaks) following DNA damage (35). Interestingly, γ-irradiation of these mice causes acute toxicity of the epithelia of their small intestines (35), as has been observed with other DNA damage and signalling and repair enzyme deficiencies (2, 3), thus emphasizing the crucial function of DNA surveillance programs of rapidly dividing cells. Similar results indicating that PARP is important for the maintenance of genomic stability following environmental or experimental stress were recently obtained (54). In this work, we have used the two-hybrid system to identify genes encoding proteins that putatively interact with PARP and are involved in its biological function. The human PARP cDNA fused to the LexA-encoding DNA-binding domain (DBD) was used as bait to screen a HeLa cDNA library fused with the activation domain of Gal4. This screening resulted in the identification of the BER pathway protein XRCC1 (X-ray repair cross-complementing 1) as a factor that associates with PARP. This interaction was further confirmed by in vivo experiments with glutathione S-transferase (GST)-tagged fusion proteins expressed in Cos-7 and HeLa cells. XRCC1 and PARP were found to interact via their respective BRCT (BRCA1 C terminus) modules (4, 9) and via an additional site located in the N-terminal zinc-finger domain of PARP. This association dramatically decreased the catalytic activity of PARP without modifying its nick sensor function. Therefore, the association of PARP with XRCC1, a partner of DNA ligase III (7, 8) and DNA polymerase β (25), is suggestive of a role in the detection and protection of a DNA strand break and the subsequent targeting of a BER complex to the damaged site.

To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vλ1 light chain genes from naive and memory B cells in DNA repair–deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand–break repair, and AAG-mediated base excision repair.

Poly(ADP-ribose) polymerase (PARP) is an element of the DNA damage surveillance network evolved by eukaryotic cells to cope with numerous environmental and endogenous genotoxic agents. PARP has been found to be involved in vivo in both cell proliferation and base excision repair of DNA. In this study the interaction between PARP and the DNA polymerase alpha-primase tetramer has been examined. We provide evidence that in proliferating cells: (i) PARP is physically associated with the catalytic subunit of the DNA polymerase alpha-primase tetramer, an association confirmed by confocal microscopy, demonstrating that both enzymes are co-localized at the nuclear periphery of HeLa cells; (ii) this interaction requires the integrity of the second zinc finger of PARP and is maximal during the S and G2/M phases of the cell cycle; (iii) PARP-deficient cells derived from PARP knock-out mice exhibited reduced DNA polymerase activity, compared with the parental cells, a reduction accentuated following exposure to sublethal doses of methylmethanesulfonate. Altogether, the present results strongly suggest that PARP participates in a DNA damage survey mechanism implying its nick-sensor function as part of the control of replication fork progression when breaks are present in the template.

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 γ-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body γ-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.

Poly(ADP-ribose) polymerase (PARP) participates in the intricate network of systems developed by the eukaryotic cell to cope with the numerous environmental and endogenous genotoxic agents. Cloning of the PARP gene has allowed the development of genetic and molecular approaches to elucidate the structure and function of this abundant and highly conserved enzyme.

The conformational changes induced by the introduction of a central and unique single-stranded break in a 139 base-pair DNA duplex have been analysed by means of polyacrylamide gel electrophoresis, HPLC and dark-field electron microscopy. Compared to the control DNA, the disruption of the covalent sugar-phosphate backbone induces a retardation detected both by gel electrophoresis and anion exchange based HPLC. Electron microscopic visualization of the DNA molecules reveals that most of them present a central fracture at the position of the nick. Measures of the angle at the apex were very well fitted by a simple model of isotropic flexible junction assuming spatial Hooke's law and simple basic Boltzmann statistics. This amounts to using a folded Gaussian distribution. The fit yields an angle equilibrium value phi 0 = 122 degrees for the nicked fragment. The angle distribution could also result from an equilibrium between two forms of the molecule with isotropic flexibility at the nicked site: a stacked and a very flexible unstacked form. The majority of bound poly(ADP-ribose) polymerase, a zinc-finger enzyme involved in DNA break detection, was localized at the apex of the V-shaped DNA duplex, with an accentuation of its general V-shaped conformation (phi 0 = 102 degrees).

A number of roles have been ascribed to poly(ADP-ribose) polymerase, (PARP; EC. 2.4.2.30), including involvement in DNA repair, cell proliferation, differentiation and transformation (1-4). One of our major goals is to understand the molecular basis of the complex mechanism leading to the PARP activation in response to DNA strand breaks. Cloning of the gene has allowed the development of molecular biological tools to elucidate the structure and the function(s) of this highly conserved enzyme. This paper describes the recent results obtained in our laboratory using these new approaches.

We describe a new vector designed to produce beta-galactosidase fusion proteins which can be used to assess subcellular localization of target peptide fragments or proteins in eukaryotic cells. The vector was constructed in such a way as to produce the peptide of interest in fusion via a short linker of proline residues to the N terminus of the reporter protein. Efficiency of the transport machinery is optimized using this particular protein fusion construction. This vector has potential uses for readily testing putative nuclear localization sequences and identifying their crucial amino-acid residues.

Poly(ADP-ribose)polymerase is a chromatin-associated enzyme of eukaryotic cell nuclei that catalyses the covalent attachment of ADP-ribose units from NAD+ to various nuclear acceptor proteins. This post-translational modification has been postulated to influence several chromatin functions, particularly those where nicking and rejoining of DNA occur. Poly(ADP-ribosyl)ation reactions are strictly dependent upon the presence of interruptions on DNA. We have recently demonstrated that the DNA-binding domain of the protein containing two putative "zinc-fingers" binds DNA in a zinc-dependent manner. The basis for the recognition of the DNA strand breaks by this enzyme, and more precisely, its 29,000 Mr N-terminal part, which contains the metal binding sites, needed to be clarified. DNA probes harbouring a single strand interruption at a defined position were constructed from synthetic oligonucleotides. DNase I protection studies show that poly(ADP-ribose)polymerase specifically binds to a DNA single-strand break by its metal-binding domain depending upon the presence of Zn(II). These results support the idea that the enzyme participates to the maintenance of DNA integrity in eukaryotes.

By Energy Dispersive X-ray fluorescence we have determined that calf thymus poly(ADP-ribose) polymerase binds two zinc ions per enzyme molecule. Using 65Zn (II) for detection of zinc binding proteins and polypeptides on western blots, we found that the zinc binding sites are localized in a 29 kd N-terminal fragment which is included in the DNA binding domain. Metal depletion and restoration experiments proved that zinc is essential for the binding of this fragment to DNA as tested by Southwestern assay. These results correlate with the existence of two putative zinc finger motifs present in the N-terminal part of the human enzyme. Poly(ADP-ribose)polymerase fingers could be involved in the recognition of DNA strand breaks and therefore in enzyme activation.

The DNA damage-dependent poly(ADP-ribose) polymerase-2 (PARP-2) is, together with PARP-1, an active player of the base excision repair process, thus defining its key role in genome surveillance and protection. Telomeres are specialized DNA-protein structures that protect chromosome ends from being recognized and processed as DNA strand breaks. In mammals, telomere protection depends on the T(2)AG(3) repeat binding protein TRF2, which has been shown to remodel telomeres into large duplex loops (t-loops). In this work we show that PARP-2 physically binds to TRF2 with high affinity. The association of both proteins requires the N-terminal domain of PARP-2 and the myb domain of TRF2. Both partners colocalize at promyelocytic leukemia bodies in immortalized telomerase-negative cells. In addition, our data show that PARP activity regulates the DNA binding activity of TRF2 via both a covalent heteromodification of the dimerization domain of TRF2 and a noncovalent binding of poly(ADP-ribose) to the myb domain of TRF2. PARP-2(-/-) primary cells show normal telomere length as well as normal telomerase activity compared to wild-type cells but display a spontaneously increased frequency of chromosome and chromatid breaks and of ends lacking detectable T(2)AG(3) repeats. Altogether, these results suggest a functional role of PARP-2 activity in the maintenance of telomere integrity.

Human papillomavirus E2 protein is a transcription factor of viral gene expression and DNA replication. Here we show that PARP is a positive regulator of the E2 protein of human papillomavirus type 18 (HPV-18). PARP interacted with the COOH terminal region of HPV-18 E2 in vitro. The E2 interaction domain within PARP is located in the NH(2)-terminal zinc finger motif and the BRCT motif included in the automodification domain. Overexpression of either wild type or the NH(2)-terminal region of PARP containing zinc finger and BRCT stimulated E2-dependent transcription. Gel retardation assay indicates that PARP augments DNA binding activity of E2 in vitro. We also show that PARP-1 is recruited to E2-dependent promoter in vivo using ChIP assay. These results suggest that PARP serves a transcriptional co-activator in E2-dependent transcription by interacting directly with the HPV E2 protein.

In eukaryote cells poly(ADP-ribose) polymerase (PARP) catalyzes the covalent attachment of ADP-ribose units from the coenzyme NAD+ to various nuclear acceptor proteins. This reaction strickly requires the presence of DNA strand breaks to which the zinc finger domain of the enzyme binds specifically (1). We have expressed a cDNA fragment encoding the two zinc fingers of human PARP (234 amino-terminal residues) in E.coli as a fusion protein. The cDNA insert was previously mutated by PCR to introduce a stop codon at its 3' end and Pstl sites at both extremities . The cDNA was then cloned in Pstl site of the E.coli expression vector pTG161 provided by M.Courtney (Transgene-Strasbourg) (2) resulting in the plasmid pTG161-FWT. Transcription of the cloned sequence proceeds from the XpL promoter which is controlled by the host encoded thermosensitive repressor cl 857. Upon a temperature shift induction from 280C (non induced) to 370C (induced) for 2 hours in LB medium, TGE 900 strain harboring the pTG161-FWT plasmid overexpressed a 35 kDa protein (13 amino-terminal residues of Xcil gene fused with the 234 first amino-acids of human PARP). This protein was easily identified in crude extracts (fig. A) by westem blot (slot 4,5), zinc blot (slot 6,7) and southwestern blot (3) (slot 8,9) .The same sonicated E.coli extracts in SDS PAGE sample buffer (Tris-HCI pH 6.8 50mM; urea 6M; 2 mercaptoethanol 6%; SDS 3% ;bromophenol blue 0.003%) were electrophoresed, transferred onto nitrocellulose membrane, renatured and processed for a "blot and footprint" experiment as described previously (1). A double stranded DNA probe harboring a strand break at a defined position was constructed from synthetic oligonucleotides(1). DNase I protection results displayed in figure B show that the finger domain of human PARP produced in E.cofi binds to the strand break and protects this region from DNasel digestion as in the case of purified calf thymus PARP (1).This simple assay which utilizes crude extracts, appears to be a valuable tool for the study of the interaction between the finger domain of PARP and a DNA strand break and could be easily generalized for the study of other finger proteins overexpressed in E.coli Fjg.A analysis of crude extracts of TGE900/pTG1 61 -FWT E.coli strain expressing the zinc finger domain of human PARP. Slots 2,4,6,8 non induced (-) and 3,5,7,9 induced (+) extracts.Fig.B DNase I footprint assay