Caspase-3-mediated processing of poly(ADP-ribose) glycohydrolase during apoptosis

Poly(ADP-ribose) glycohydrolase (PARG) is responsible for the catabolism of poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerase (PARP-1) and other PARP-1-like enzymes. In this work, we report that PARG is cleaved during etoposide-, staurosporine-, and Fas-induced apoptosis in human cells. This cleavage is concomitant with PARP-1 processing and generates two C-terminal fragments of 85 and 74 kDa. In vitro cleavage assays using apoptotic cell extracts showed that a protease of the caspase family is responsible for PARG processing. A complete inhibition of this cleavage was achieved at nanomolar concentrations of the caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting the involvement of caspase-3-like proteases. Consistently, recombinant caspase-3 efficiently cleaved PARG in vitro, suggesting the involvement of this protease in PARG processing in vivo. Furthermore, caspase-3-deficient MCF-7 cells did not show any PARG cleavage in response to staurosporine treatment. The cleavage sites identified by site-directed mutagenesis are DEID(256) downward arrow V and the unconventional site MDVD(307) downward arrow N. Kinetic studies have shown similar maximal velocity (V(max)) and affinity (K(m)) for both full-length PARG and its apoptotic fragments, suggesting that caspase-3 may affect PARG function without altering its enzymatic activity. The early cleavage of both PARP-1 and PARG by caspases during apoptosis suggests an important function for poly(ADP-ribose) metabolism regulation during this cell death process.     

Poly(ADP-ribose) polymerase 1 accelerates single-strand break repair in concert with poly(ADP-ribose) glycohydrolase

Single-strand breaks are the commonest lesions arising in cells, and defects in their repair are implicated in neurodegenerative disease. One of the earliest events during single-strand break repair (SSBR) is the rapid synthesis of poly(ADP-ribose) (PAR) by poly(ADP-ribose) polymerase (PARP), followed by its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). While the synthesis of poly(ADP-ribose) is important for rapid rates of chromosomal SSBR, the relative importance of poly(ADP-ribose) polymerase 1 (PARP-1) and PARP-2 and of the subsequent degradation of PAR by PARG is unclear. Here we have quantified SSBR rates in human A549 cells depleted of PARP-1, PARP-2, and PARG, both separately and in combination. We report that whereas PARP-1 is critical for rapid global rates of SSBR in human A549 cells, depletion of PARP-2 has only a minor impact, even in the presence of depleted levels of PARP-1. Moreover, we identify PARG as a novel and critical component of SSBR that accelerates this process in concert with PARP-1.     

Altered poly(ADP-ribose) metabolism impairs cellular responses to genotoxic stress in a hypomorphic mutant of poly(ADP-ribose) glycohydrolase

Genotoxic stress activates nuclear poly(ADP-ribose) (PAR) metabolism leading to PAR synthesis catalyzed by DNA damage activated poly(ADP-ribose) polymerases (PARPs) and rapid PAR turnover by action of nuclear poly(ADP-ribose) glycohydrolase (PARG). The involvement of PARP-1 and PARP-2 in responses to DNA damage has been well studied but the involvement of nuclear PARG is less well understood. To gain insights into the function of nuclear PARG in DNA damage responses, we have quantitatively studied PAR metabolism in cells derived from a hypomorphic mutant mouse model in which exons 2 and 3 of the PARG gene have been deleted (PARG-Delta2,3 cells), resulting in a nuclear PARG containing a catalytic domain but lacking the N-terminal region (A domain) of the protein. Following DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we found that the activity of both PARG and PARPs in intact cells is increased in PARG-Delta2,3 cells. The increased PARG activity leads to decreased PARP-1 automodification with resulting increased PARP activity. The degree of PARG activation is greater than PARP, resulting in decreased PAR accumulation. Following MNNG treatment, PARG-Delta2,3 cells show reduced formation of XRCC1 foci, delayed H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. Our results show that a precise coordination of PARPs and PARG activities is important for normal cellular responses to DNA damage and that this coordination is defective in the absence of the PARG A domain.     

MNNG-induced cell death is controlled by interactions between PARP-1, poly(ADP-ribose) glycohydrolase, and XRCC1

PARP-1 (poly(ADP-ribose) polymerases) modifies proteins with poly(ADP-ribose), which is an important signal for genomic stability. ADP-ribose polymers also mediate cell death and are degraded by poly(ADP-ribose) glycohydrolase (PARG). Here we show that the catalytic domain of PARG interacts with the automodification domain of PARP-1. Furthermore, PARG can directly down-regulate PARP-1 activity. PARG also interacts with XRCC1, a DNA repair factor that is recruited by DNA damage-activated PARP-1. We investigated the role of XRCC1 in cell death after treatment with supralethal doses of the alkylating agent MNNG. Only in XRCC1-proficient cells MNNG induced a considerable accumulation of poly(ADP-ribose). Similarly, extracts of XRCC1-deficient cells produced large ADP-ribose polymers if supplemented with XRCC1. Consequently, MNNG triggered in XRCC1-proficient cells the translocation of the apoptosis inducing factor from mitochondria to the nucleus followed by caspase-independent cell death. In XRCC1-deficient cells, the same MNNG treatment caused non-apoptotic cell death without accumulation of poly(ADP-ribose). Thus, XRCC1 seems to be involved in regulating a poly(ADP-ribose)-mediated apoptotic cell death.     

Depletion of the 110-kilodalton isoform of poly(ADP-ribose) glycohydrolase increases sensitivity to genotoxic and endotoxic stress in mice

Poly(ADP-ribosylation) is rapidly stimulated in cells following DNA damage. This posttranslational modification is regulated by the synthesizing enzyme poly(ADP-ribose) polymerase 1 (PARP-1) and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG). Although the role of PARP-1 in response to DNA damage has been studied extensively, the function of PARG and the impact of poly(ADP-ribose) homeostasis in various cellular processes are largely unknown. Here we show that by gene targeting in embryonic stem cells and mice, we specifically deleted the 110-kDa PARG protein (PARG(110)) normally found in the nucleus and that depletion of PARG(110) severely compromised the automodification of PARP-1 in vivo. PARG(110)-deficient mice were viable and fertile, but these mice were hypersensitive to alkylating agents and ionizing radiation. In addition, these mice were susceptible to streptozotocin-induced diabetes and endotoxic shock. These data indicate that PARG(110) plays an important role in DNA damage responses and in pathological processes.     

Poly(ADP-ribose) degradation by post-nuclear extracts from human cells

The nuclear metabolism of poly(ADP-ribose) is mainly regulated by poly(ADP-ribose) polymerase-1 (PARP-1) and by poly(ADP-ribose) glycohydrolase (PARG). A PARP-like enzyme, V-PARP, and a PARG isoform are present in the extra-nuclear compartment of mammalian cells, even if poly(ADP-ribose) has never been detected therein. In this work, we demonstrate the ability of post-nuclear extracts from HeLa and HL60 cells to degrade synthetic 32P-polymers of ADP-ribose to ADP-ribose and, further, to AMP. This reaction implies the combined action of PARG and of an ADP-ribose-degrading activity, possibly corresponding to a phosphodiesterase and/or to an ADP-ribose pyrophosphatase. The inhibition of PARG or ADP-ribose-degrading enzymes allowed the demonstration that in vitro synthesized 32P-poly(ADP-ribose) is first digested to ADP-ribose monomers by a typical PARG reaction, and that ADP-ribose is further rapidly converted into AMP by an Mg(2+)-dependent activity. Collectively, our results demonstrate the ability of the human cell post-nuclear fraction to convert synthetic poly(ADP-ribose) into utilizable AMP units by the concerted action of PARG and ADP-ribose-degrading activities.     

Herpes simplex virus 1 infection activates poly(ADP-ribose) polymerase and triggers the degradation of poly(ADP-ribose) glycohydrolase

Herpes simplex virus 1 infection triggers multiple changes in the metabolism of host cells, including a dramatic decrease in the levels of NAD(+). In addition to its role as a cofactor in reduction-oxidation reactions, NAD(+) is required for certain posttranslational modifications. Members of the poly(ADP-ribose) polymerase (PARP) family of enzymes are major consumers of NAD(+), which they utilize to form poly(ADP-ribose) (PAR) chains on protein substrates in response to DNA damage. PAR chains can subsequently be removed by the enzyme poly(ADP-ribose) glycohydrolase (PARG). We report here that the HSV-1 infection-induced drop in NAD(+) levels required viral DNA replication, was associated with an increase in protein poly(ADP-ribosyl)ation (PARylation), and was blocked by pharmacological inhibition of PARP-1/PARP-2 (PARP-1/2). Neither virus yield nor the cellular metabolic reprogramming observed during HSV-1 infection was altered by the rescue or further depletion of NAD(+) levels. Expression of the viral protein ICP0, which possesses E3 ubiquitin ligase activity, was both necessary and sufficient for the degradation of the 111-kDa PARG isoform. This work demonstrates that HSV-1 infection results in changes to NAD(+) metabolism by PARP-1/2 and PARG, and as PAR chain accumulation can induce caspase-independent apoptosis, we speculate that the decrease in PARG levels enhances the auto-PARylation-mediated inhibition of PARP, thereby avoiding premature death of the infected cell.     

Role of poly(ADP-ribose) glycohydrolase in the development of inflammatory bowel disease in mice

Poly(ADP-ribose) is synthesized from nicotinamide adenine dinucleotide (NAD) by poly(ADP-ribose) polymerase 1 (PARP-1) and degraded by poly(ADP-ribose) glycohydrolase (PARG). The aim of the present study was to examine the role of PARG in the development of experimental colitis. To address this question, we used an experimental model of colitis, induced by dinitrobenzene sulfonic acid (DNBS). Mice lacking the functional 110-kDa isoform of PARG (PARG(110)KO mice) were resistant to colon injury induced by DNBS. The mucosa of colon tissues showed reduction of myeloperoxidase activity and attenuated staining for intercellular adhesion molecule 1 and vascular cell adhesion molecule 1. Moreover, overproduction of proinflammatory factors TNF-alpha and IL-1beta and activation of cell death signaling pathway, i.e., the FAS ligand, were inhibited in these mutant mice. Finally pharmacological treatment of WT mice with GPI 16552 and 18214, two novel PARG inhibitors, showed a significant protective effect in DNBS-induced colitis. These genetic and pharmacological studies demonstrate that PARG modulates the inflammatory response and tissue injury events associated with colitis and PARG may be considered as a novel target for pharmacological intervention for the pathogenesis.     

Poly(ADP-ribose) polymerases: managing genome stability

The importance of poly(ADP-ribose) metabolism in the maintenance of genomic integrity following genotoxic stress has long been firmly established. Poly(ADP-ribose) polymerase-1 (PARP-1) and its catabolic counterpart, poly(ADP-ribose) glycohydrolase (PARG) play major roles in the modulation of cell responses to genotoxic stress. Recent discoveries of a number of other enzymes with poly(ADP-ribose) polymerase activity have established poly(ADP-ribosyl)ation as a general biological mechanism in higher eukaryotic cells that not only promotes cellular recovery from genotoxic stress and eliminates severely damaged cells from the organism, but also ensures accurate transmission of genetic information during cell division. Additionally, emerging data suggest the involvement of poly(ADP-ribosyl)ation in the regulation of intracellular trafficking, memory formation and other cellular functions. In this brief review on PARP and PARG enzymes, emphasis is placed on PARP-1, the best understood member of the PARP family and on the relationship of poly(ADP-ribosyl)ation to cancer and other diseases of aging.     

Global Analysis of Transcriptional Regulation by Poly(ADP-ribose) Polymerase-1 and Poly(ADP-ribose) Glycohydrolase in MCF-7 Human Breast Cancer Cells

Poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes that modify target proteins by the addition and removal, respectively, of ADP-ribose polymers. Although a role for PARP-1 in gene regulation has been well established, the role of PARG is less clear. To investigate how PARP-1 and PARG coordinately regulate global patterns of gene expression, we used short hairpin RNAs to stably knock down PARP-1 or PARG in MCF-7 cells followed by expression microarray analyses. Correlation analyses showed that the majority of genes affected by the knockdown of one factor were similarly affected by the knockdown of the other factor. The most robustly regulated common genes were enriched for stress-response and metabolic functions. In chromatin immunoprecipitation assays, PARP-1 and PARG localized to the promoters of positively and negatively regulated target genes. The levels of chromatin-bound PARG at a given promoter generally correlated with the levels of PARP-1 across the subset of promoters tested. For about half of the genes tested, the binding of PARP-1 at the promoter was dependent on the binding of PARG. Experiments using stable re-expression of short hairpin RNA-resistant catalytic mutants showed that PARP-1 and PARG enzymatic activities are required for some, but not all, target genes. Collectively, our results indicate that PARP-1 and PARG, nuclear enzymes with opposing enzymatic activities, localize to target promoters and act in a similar, rather than antagonistic, manner to regulate gene expression.     

Poly(ADP-ribose) protects vascular smooth muscle cells from oxidative DNA damage

Vascular smooth muscle cells (VSMCs) undergo death during atherosclerosis, a widespread cardiovascular disease. Recent studies suggest that oxidative damage occurs in VSMCs and induces atherosclerosis. Here, we analyzed oxidative damage repair in VSMCs and found that VSMCs are hypersensitive to oxidative damage. Further analysis showed that oxidative damage repair in VSMCs is suppressed by a low level of poly (ADP-ribosyl)ation (PARylation), a key post-translational modification in oxidative damage repair. The low level of PARylation is not caused by the lack of PARP-1, the major poly(ADP-ribose) polymerase activated by oxidative damage. Instead, the expression of poly(ADP-ribose) glycohydrolase, PARG, the enzyme hydrolyzing poly(ADP-ribose), is significantly higher in VSMCs than that in the control cells. Using PARG inhibitor to suppress PARG activity facilitates oxidative damage-induced PARylation as well as DNA damage repair. Thus, our study demonstrates a novel molecular mechanism for oxidative damage-induced VSMCs death. This study also identifies the use of PARG inhibitors as a potential treatment for atherosclerosis. [BMB Reports 2015; 48(6): 354-359]     

Tannins elevate the level of poly(ADP-ribose) in HeLa cell extracts

Phenolic phytochemicals such as tannins, which are natural constituents of green tea, red wine, and other plant products, are considered to have cancer-preventive properties. An important endogenous mediator of tumorigenesis is the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 synthesizes polymers of ADP-ribose (PAR), which, in turn, are degraded by the catabolic enzyme poly(ADP-ribose) glycohydrolase (PARG). In the present study, we investigated the effects of tannins on the level of PAR in HeLa nuclear extracts. The addition of tannins to nuclear extracts led to a 40-fold elevation of PAR-levels. The observed increased PAR-levels resulted from inhibition of the catalytic activity of PARG. Additionally, the human PARG cDNA was cloned and the recombinant enzyme was overexpressed and isolated. Recombinant PARG was immobilized using an affinity column composed of tannins covalently linked to Sepharose beads. Finally, an interaction between immobilized PARG and endogenous PARP-1 from HeLa cell extracts is demonstrated.     

Alteration of poly(ADP-ribose) glycohydrolase nucleocytoplasmic shuttling characteristics upon cleavage by apoptotic proteases

Poly(ADP-ribosyl)ation is an important post-translational modification which mostly affects nuclear proteins. The major roles of poly(ADP-ribose) synthesis are assigned to DNA damage signalling during base excision repair, apoptosis and excitotoxicity. The transient nature and modulation of poly(ADP-ribose) levels depend mainly on the activity of poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG), the key catabolic enzyme of poly(ADP-ribose). Given the fact that PARG substrate, poly(ADP-ribose), is found almost exclusively in the nucleus and that PARG is mainly localized in the cytoplasm, we wanted to have a closer look at PARG subcellular localization in order to better understand the mechanism by which PARG regulates intracellular poly(ADP-ribose) levels. We examined the subcellular distribution of PARG and of its two enzymatically active C-terminal apoptotic fragments both biochemically and by fluorescence microscopy. Green fluorescent protein (GFP) fusion proteins were constructed for PARG (GFP-PARG), its 74 kDa (GFP-74) and 85 kDa (GFP-85) apoptotic fragments and transiently expressed in COS-7 cells. Localization experiments reveal that all three fusion proteins localize predominantly to the cytoplasm and that a fraction also co-localizes with the Golgi marker FTCD. Moreover, leptomycin B, a drug that specifically inhibits nuclear export signal (NES)-dependent nuclear export, induces a redistribution of GFP-PARG from the cytoplasm to the nucleus and this nuclear accumulation is even more pronounced for the GFP-74 and GFP-85 apoptotic fragments. This observation confirms our hypothesis for the presence of important regions in the PARG sequence that would allow the protein to engage in CRM1-dependent nuclear export. Moreover, the altered nuclear import kinetics found for the apoptotic fragments highlights the importance of PARG N-terminal sequence in modulating PARG nucleocytoplasmic trafficking properties.     

Importance of poly(ADP-ribose) glycohydrolase in the control of poly(ADP-ribose) metabolism.

Poly(ADP-ribosyl)ation is a posttranslational modification that alters the functions of the acceptor proteins and is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes. Following DNA damage, activated poly(ADP-ribose) polymerase-1 (PARP-1) catalyzes the elongation and branching of poly(ADP-ribose) (pADPr) covalently attached to nuclear target proteins. Although the biological role of poly(ADP-ribosyl)ation has not yet been defined, it has been implicated in many important cellular processes such as DNA repair and replication, modulation of chromatin structure, and apoptosis. The transient nature and modulation of poly(ADP-ribosyl)ation depend on the activity of a unique cytoplasmic enzyme called poly(ADP-ribose) glycohydrolase which hydrolyzes pADPr bound to acceptor proteins in free ADP-ribose residues. While the PARP homologues have been recently reviewed, there are relatively scarce data about PARG in the literature. Here we summarize the latest advances in the PARG field, addressing the question of its putative nucleo-cytoplasmic shuttling that could enable the tight regulation of pADPr metabolism. This would contribute to the elucidation of the biological significance of poly(ADP-ribosyl)ation.     

PARG is recruited to DNA damage sites through poly(ADP-ribose)- and PCNA-dependent mechanisms

Post-translational poly(ADP-ribosyl)ation has diverse essential functions in the cellular response to DNA damage as it contributes to avid DNA damage detection and assembly of the cellular repair machinery but extensive modification eventually also induces cell death. While there are 17 human poly(ADP-ribose) polymerase (PARP) genes, there is only one poly(ADP-ribose) glycohydrolase (PARG) gene encoding several PARG isoforms located in different subcellular compartments. To investigate the recruitment of PARG isoforms to DNA repair sites we locally introduced DNA damage by laser microirradiation. All PARG isoforms were recruited to DNA damage sites except for a mitochondrial localized PARG fragment. Using PARP knock out cells and PARP inhibitors, we showed that PARG recruitment was only partially dependent on PARP-1 and PAR synthesis, indicating a second, PAR-independent recruitment mechanism. We found that PARG interacts with PCNA, mapped a PCNA binding site and showed that binding to PCNA contributes to PARG recruitment to DNA damage sites. This dual recruitment mode of the only nuclear PARG via the versatile loading platform PCNA and by a PAR dependent mechanism likely contributes to the dynamic regulation of this posttranslational modification and ensures the tight control of the switch between efficient DNA repair and cell death.     

Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V_max) and the michaelis constant (k_m) of PARG reaction were 4.46 μM and 128.33 μmol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 μM. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.     

Bacterial production of recombinant human poly(ADP-ribose) glycohydrolase

Poly(ADP-ribosyl)ation, which is mainly involved in DNA repair and replication, is catalyzed mainly by poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Although recombinant human PARP-1 (hPARP-1) is commercially available, there are no reports on the preparation of recombinant human PARG (hPARG). Here, we report the efficient expression and purification of a recombinant hPARG-catalytic domain (hPARG-CD) from Escherichia coli (E. coli). hPARG-CD was expressed as a fusion protein with a glutathione S-transferase (GST) tag at the N-terminus and a hexahistidine (6His) tag at the C-terminus. Both high cell density and low temperature culture conditions were important for the maximum production of soluble recombinant hPARG-CD. After sequential affinity chromatography using immobilized metal affinity resin and glutathione-Sepharose (GSH-Sephasrose), more than 95% pure recombinant hPARG-CD was obtained with a yield of approximately 2mg per 1L of E. coli culture medium. The km and Vmax values of purified recombinant hPARG-CD were 9.0 μM and 35.6 μmol/min/mg protein, respectively. These kinetic values were similar to those of purified endogenous hPARG reported previously. Furthermore, the recombinant hPARG-CD was inhibited by known PARG inhibitors such as adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), eosin Y, and phloxine B. These results show that the recombinant hPARG-CD is useful to search for specific inhibitors and to elucidate the regulatory mechanisms of hPARG.     

Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update

Poly(ADP-ribosylation) is a post-translational modification of proteins playing a crucial role in many processes, including DNA repair and cell death. The best known poly(ADP-ribosylating) enzime, PARP-1, is a DNA nick sensor and uses NAD⁺ to form polymers of ADP-ribose which are further bound to nuclear protein acceptors. To strictly regulate poly(ADP-ribose) turnover, its degradation is assured by the enzyme poly(ADP-ribose) glycohydrolase (PARG). During apoptosis, PARP-1 plays two opposite roles: its stimulation leads to poly(ADP-ribose) synthesis, whereas caspases cause PARP-1 cleavage and inactivation. PARP-1 proteolysis produces an 89 kDa C-terminal fragment, with a reduced catalytic activity, and a 24 kDa N-terminal peptide, which retains the DNA binding domains. The fate and the possible role of these fragments during apoptosis will be discussed.     

Inhibition of poly(ADP-ribose) glycohydrolase (PARG) specifically kills BRCA2-deficient tumor cells

Poly(ADP-ribose) glycohydrolase (PARG), removes poly(ADP-ribose) subunits from proteins that have previously been modified by poly(ADP-ribose) polymerse. This ensures that modification is transient, and it is suggested that removal of poly(ADP-ribose) is essential for some types of DNA repair. Here we show increased γH2AX foci formation and increased homologous recombination when PARG is inhibited. These effects are reduced when replication is inhibited, suggesting that in the absence of PARG activity, replication forks collapse, and homologous recombination is induced for repair. Consistent with this, we show that cells deficient in the homologous recombination protein BRCA2 are sensitive to PARG depletion or inhibition. These data raise the exciting possibility that PARG inhibitors may be used to specifically kill BRCA2 and other homologous recombination-deficient tumors.     

Dynamic relocation of poly(ADP-ribose) glycohydrolase isoforms during radiation-induced DNA damage

Poly(ADP-ribosyl)ation is a very early cellular response to DNA damage. Poly(ADP-ribose) (PAR) accumulation is transient since PAR is rapidly hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG). PARG may play a prominent role in DNA damage response and repair by removing PAR from modified proteins including PARP-1. Using living cells, we provide evidence that in response to DNA damage induced by gamma-irradiation the cytoplasmic 103 kDa PARG isoform translocates into the nucleus. We further observed that the nuclear GFP-hPARG110 enzyme relocalizes to the cytoplasm in response to DNA damage. Using different GFP-PARG fusion proteins specific for the nuclear and cytoplasmic forms, we demonstrate their dynamic distribution between cytoplasm and nucleoplasm and a high mobility of major PARG isoforms by fluorescence recovery after photobleaching (FRAP). The dynamic relocation of all PARG isoforms presented in this report reveals a novel biological mechanism by which PARG could be involved in DNA damage response.