Purification of Recombinant Human PARG and Activity Assays.

The purification of poly(ADP-ribose) glycohydrolase (PARG) from overexpressing bacteria Escherichia coli is described here as a fast and reproducible one chromatographic step protocol. After cell lysis, GST-PARG-fusion proteins from the crude extract are affinity purified by a glutathione 4B sepharose chromatographic step. The PARG proteins are then freed from their GST-fusion by overnight enzymatic cleavage using the preScission protease. As described in the protocol, more than 500 µg of highly active human PARG can be obtained from 1.5 L of E. coli culture.

Purification of Recombinant Human PARP-3.

The purification of poly(ADP-ribose) polymerase-3 (PARP-3) from overexpressing cells (Sf9 insect cells, Escherichia coli) has been updated to a fast and reproducible two-chromatographic-step protocol. After cell lysis, PARP-3 protein from the crude extract is affinity purified on a 3-aminobenzamide Sepharose™ chromatographic step. The last contaminants and the 3-methoxybenzamide used to elute PARP-3 from the previous affinity column are removed on the high-performance strong cation exchanger MonoQ™ matrix. This step allows also the concentration of the protein. The columns connected to an A° KTA™ purifier system allow the purification of the protein in three days with a high-yield recovery. As described in the protocol, more than 3 mg of pure and active human PARP-3 can be obtained from 1.5 L of E. coli culture.

PARP3 supervises G9a-mediated repression of adhesion and hypoxia-responsive genes in glioblastoma cells.

In breast cancer, Poly(ADP-ribose) polymerase 3 (PARP3) has been identified as a key driver of tumor aggressiveness exemplifying its selective inhibition as a promising surrogate for clinical activity onto difficult-to-treat cancers. Here we explored the role of PARP3 in the oncogenicity of glioblastoma, the most aggressive type of brain cancer. The absence of PARP3 did not alter cell proliferation nor the in vivo tumorigenic potential of glioblastoma cells. We identified a physical and functional interaction of PARP3 with the histone H3 lysine 9 methyltransferase G9a. We show that PARP3 helps to adjust G9a-dependent repression of the adhesion genes Nfasc and Parvb and the hypoxia-responsive genes Hif-2α, Runx3, Mlh1, Ndrg1, Ndrg2 and Ndrg4. Specifically for Nfasc, Parvb and Ndrg4, PARP3/G9a cooperate for an adjusted establishment of the repressive mark H3K9me2. While examining the functional consequence in cell response to hypoxia, we discovered that PARP3 acts to maintain the cytoskeletal microtubule stability. As a result, the absence of PARP3 markedly increases the sensitivity of glioblastoma cells to microtubule-destabilizing agents providing a new therapeutic avenue for PARP3 inhibition in brain cancer therapy.

Gemcitabine-Based Chemoradiotherapy Enhanced by a PARP Inhibitor in Pancreatic Cancer Cell Lines.

Pancreatic ductal adenocarcinoma is a devastating disease with a 5-year overall survival of 9% for all stages. Gemcitabine-based chemoradiotherapy for locally advanced pancreatic cancer is highly toxic. We conducted an in vitro study to determine whether poly(ADP-ribose) polymerase-1 inhibition radiosensitized gemcitabine-based chemotherapy. Human pancreatic cancer cell lines, MIA PaCa-2, AsPC-1, BxPC-3 and PANC-1 were treated with gemcitabine (10 nM) and/or olaparib (1 µM). Low-LET gamma single dose of 2, 5 and 10 Gy radiations were carried out. Clonogenic assay, PAR immunoblotting, cell cycle distribution, γH2Ax, necrotic and autophagic cell death quantifications were performed. Treatment with olaparib alone was not cytotoxic, but highly radiosensitized cell lines, particularly at high dose per fraction A non-cytotoxic concentration of gemcitabine radiosensitized cells, but less than olaparib. Interestingly, olaparib significantly enhanced gemcitabine-based radiosensitization in PDAC cell lines with synergistic effect in BxPC-3 cell line. All cell lines were radiosensitized by the combination of gemcitabine and olaparib, through an increase of unrepaired double-strand, a G2 phase block and cell death. Radiosensitization was increased with high dose of radiation. The combination of olaparib with gemcitabine-based chemoradiotherapy could lead to an enhancement of local control in vivo and an improvement in disease-free survival.

Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions.

BACKGROUND: The adaptation to hypoxia is mainly controlled by the HIF transcription factors. Increased expression/activity of HIF-1α correlates with poor prognosis in cancer patients. PARP-1 inhibitors are used in the clinic to treat BRCAness breast/ovarian cancer and have been shown to regulate the hypoxic response; therefore, their use could be expanded. METHODS: In this work by integrating molecular/cell biology approaches, genome-wide ChIP-seq, and patient samples, we elucidate the extent to which PARP-1 exerts control over HIF-1-regulated genes. RESULTS: In human melanoma, PARP-1 and HIF-1α expression are strongly associated. In response to a hypoxic challenge poly(ADP-ribose) (PAR) is synthesized, HIF-1α is post-transcriptionally modified (PTM) and stabilized by PARylation at specific K/R residues located at its C-terminus. Using an unbiased ChIP-seq approach we demonstrate that PARP-1 dictates hypoxia-dependent HIF-recruitment to chromatin in a range of HIF-regulated genes while analysis of HIF-binding motifs (RCGTG) reveals a restriction on the recognition of hypoxia responsive elements in the absence of PARP-1. Consequently, the cells are poorly adapted to hypoxia, showing a reduced fitness during hypoxic induction. CONCLUSIONS: These data characterize the fine-tuning regulation by PARP-1/PARylation of HIF activation and suggest that PARP inhibitors might have therapeutic potential against cancer types displaying HIF-1α over-activation.

Parp3 promotes astrocytic differentiation through a tight regulation of Nox4-induced ROS and mTorc2 activation.

Parp3 is a member of the Poly(ADP-ribose) polymerase (Parp) family that has been characterized for its functions in strand break repair, chromosomal rearrangements, mitotic segregation and tumor aggressiveness. Yet its physiological implications remain unknown. Here we report a central function of Parp3 in the regulation of redox homeostasis in continuous neurogenesis in mice. We show that the absence of Parp3 provokes Nox4-induced oxidative stress and defective mTorc2 activation leading to inefficient differentiation of post-natal neural stem/progenitor cells to astrocytes. The accumulation of ROS contributes to the decreased activity of mTorc2 as a result of an oxidation-induced and Fbxw7-mediated ubiquitination and degradation of Rictor. In vivo, mTorc2 signaling is compromised in the striatum of naïve post-natal Parp3-deficient mice and 6 h after acute hypoxia-ischemia. These findings reveal a physiological function of Parp3 in the tight regulation of striatal oxidative stress and mTorc2 during astrocytic differentiation and in the acute phase of hypoxia-ischemia.

Purification of Recombinant Human PARP-3.

The purification of poly(ADP-ribose) polymerase-3 (PARP-3) from overexpressing cells (Sf9 insect cells, Escherichia coli) has been updated to a fast and reproducible two chromatographic steps protocol. After cell lysis, PARP-3 protein from the crude extract is affinity purified on a 3-aminobenzamide Sepharose™ chromatographic step. The last contaminants and the 3-methoxybenzamide used to elute PARP-3 from the previous affinity column are removed on the high-performance strong cations exchanger MonoQ™ matrix. This step allows also the concentration of the protein. The columns connected to an ÅKTA™ purifier system allow the purification of the protein in 3 days with a high-yield recovery. As described in the protocol, more than 3 mg of pure and active human PARP-3 can be obtained from 1.5 L of E. coli culture.

Purification of Recombinant Human PARG and Activity Assays.

The purification of Poly(ADP-ribose) glycohydrolase (PARG) from overexpressing bacteria Escherichia coli is described here to a fast and reproducible one chromatographic step protocol. After cell lysis, GST-PARG-fusion proteins from the crude extract are affinity purified by a Glutathione 4B Sepharose chromatographic step. The PARG proteins are then freed from their GST-fusion by overnight enzymatic cleavage using the preScission protease. As described in the protocol, more than 500 µg of highly active human PARG can be obtained from 1.5 L of E. coli culture.

PARG deficiency is neither synthetic lethal with BRCA1 nor PTEN deficiency.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) inhibitors have entered the clinics for their promising anticancer effect as adjuvant in chemo- and radiotherapy and as single agent on BRCA-mutated tumours. Poly(ADP-ribose) glycohydrolase (PARG) deficiency was also shown to potentiate the cytotoxicity of genotoxic agents and irradiation. The aim of this study is to investigate the effect of PARG deficiency on BRCA1- and/or PTEN-deficient tumour cells. METHODS: Since no PARG inhibitors are available for in vivo studies, PARG was depleted by siRNA in several cancer cell lines, proficient or deficient for BRCA1 and/or PTEN. The impact on cell survival was evaluated by colony formation assay and short-term viability assays. The effect of simultaneous PARG and BRCA1 depletion on homologous recombination (HR) efficacy was evaluated by immunodetection of RAD51 foci and using an in vivo HR assay. RESULTS: The BRCA1-deficient cell lines MDA-MB-436, HCC1937 and UWB1.289 showed mild sensitivity to PARG depletion, whereas no sensitivity was observed for the BRCA1-proficient MDA-MB-231, MDA-MB-468, MCF10A and U2OS cell lines. However, the BRCA1-reconstituted UWB1.289 cell lines was similarly sensitive to PARG depletion than the BRCA1-deficient UWB1.289, and the simultaneous depletion of PARG and BRCA1 and/or PTEN in MDA-MB-231 or U2OS cells was not more cytotoxic than depletion of BRCA1 or PTEN only. CONCLUSIONS: Some tumour cells displayed slight sensitivity to PARG deficiency, but this sensitivity could not be correlated to BRCA1- or PTEN-deficiency. Therefore, PARG depletion cannot be considered as a strategy to kill tumours cells mutated in BRCA1 or PTEN.

Interaction of PARP-2 with AP site containing DNA.

In eukaryotes the stability of genome is provided by functioning of DNA repair systems. One of the main DNA repair pathways in eukaryotes is the base excision repair (BER). This system requires precise regulation for correct functioning. Two members of the PARP family - PARP-1 and PARP-2, which can be activated by DNA damage - are widely considered as regulators of DNA repair processes, including BER. In contrast to PARP-1, the role of PARP-2 in BER has not been extensively studied yet. Since AP site is one of the most frequent type of DNA damage and a key intermediate of BER at the stage preceding formation of DNA breaks, in this paper we focused on the characterization of PARP-2 interaction with AP site-containing DNAs. We demonstrated that PARP-2, like PARP-1, can interact with the intact AP site via Schiff base formation, in spite of crucial difference in the structure of the DNA binding domains of these PARPs. By cross-linking of PARPs to AP DNA, we determined that the N-terminal domains of both PARPs are involved in formation of cross-links with AP DNA. We have also confirmed that DNA binding by PARP-2, in contrast to PARP-1, is not modulated by autoPARylation. PARP-2, like PARP-1, can inhibit the activity of APE1 by binding to AP site, but, in contrast to PARP-1, this inhibitory influence is hardly regulated by PAR synthesis. At the same time, 5'-dRP lyase activity of both PARPs is comparable, although being much weaker than that of Pol ß, which is considered as the main 5'-dRP lyase of the BER process.

PARG is dispensable for recovery from transient replicative stress but required to prevent detrimental accumulation of poly(ADP-ribose) upon prolonged replicative stress.

Poly(ADP-ribosyl)ation is involved in numerous bio-logical processes including DNA repair, transcription and cell death. Cellular levels of poly(ADP-ribose) (PAR) are regulated by PAR polymerases (PARPs) and the degrading enzyme PAR glycohydrolase (PARG), controlling the cell fate decision between life and death in response to DNA damage. Replication stress is a source of DNA damage, leading to transient stalling of replication forks or to their collapse followed by the generation of double-strand breaks (DSB). The involvement of PARP-1 in replicative stress response has been described, whereas the consequences of a deregulated PAR catabolism are not yet well established. Here, we show that PARG-deprived cells showed an enhanced sensitivity to the replication inhibitor hydroxyurea. PARG is dispensable to recover from transient replicative stress but is necessary to avoid massive PAR production upon prolonged replicative stress, conditions leading to fork collapse and DSB. Extensive PAR accumulation impairs replication protein A association with collapsed forks resulting in compromised DSB repair via homologous recombination. Our results highlight the critical role of PARG in tightly controlling PAR levels produced upon genotoxic stress to prevent the detrimental effects of PAR over-accumulation.

PARP1-TDP1 coupling for the repair of topoisomerase I-induced DNA damage.

Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3'-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1-PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1.

Interaction of PARP-2 with DNA structures mimicking DNA repair intermediates and consequences on activity of base excision repair proteins.

Poly(ADP-ribosyl)ation is a posttranslational protein modification significant for genomic stability and cell survival in response to DNA damage. Poly(ADP-ribosyl)ation is catalyzed by poly(ADP-ribose)polymerases (PARPs). Among the 17 members of the PARP family, PARP-1 and PARP-2 are described as enzymes whose catalytic activity is stimulated by some types of DNA damages. Whereas the role of PARP-1 in response to DNA damage has been widely illustrated, the contribution of another DNA-dependent PARP, PARP-2, is less documented. To find out specific DNA targets of PARP-2 we evaluated by EMSA Kd values of PARP-2-DNA complexes for several DNA structures mimicking intermediates of different DNA metabolizing processes. In addition, we tested these DNA as activators of PARP-1 and PARP-2 in poly(ADP-ribose) synthesis. Like PARP-1, PARP-2 doesn't show correlation between activation efficiency and Kd values for DNA. PARP-2 displayed the highest affinity for flap-containing DNA, but was more efficiently activated by 5'-overhang DNA. Evaluating the influence of PARP-1 and PARP-2 on DNA repair synthesis catalyzed by DNA polymerase ß revealed that both PARPs inhibit DNA polymerase ß activity. However, unlike PARP-1, poly(ADP-ribosyl)ation of PARP-2 does not result in restoration of DNA synthesis efficiency. Similarly, both PARPs proteins inhibited FEN1 activity, but only activation of PARP-1, not PARP-2, could restore FEN1 activity, and only when PARP-2 was not present. Taken together, our data show that PARP-2 can directly regulate BER proteins but also can modulate the influence of PARP-1 on these BER proteins, by decreasing its poly(ADP-ribosyl)ation activity.

Poly (ADP-ribose) glycohydrolase regulates retinoic acid receptor-mediated gene expression.

Poly-(ADP-ribose) glycohydrolase (PARG) is a catabolic enzyme that cleaves ADP-ribose polymers synthesized by poly-(ADP-ribose) polymerases. Here, transcriptome profiling and differentiation assay revealed a requirement of PARG for retinoic acid receptor (RAR)-mediated transcription. Mechanistically, PARG accumulates early at promoters of RAR-responsive genes upon retinoic acid treatment to promote the formation of an appropriate chromatin environment suitable for transcription. Silencing of PARG or knockout of its enzymatic activity maintains the H3K9me2 mark at the promoter of the RAR-dependent genes, leading to the absence of preinitiation complex formation. In the absence of PARG, we found that the H3K9 demethylase KDM4D/JMJD2D became PARsylated. Mutation of two glutamic acids located in the Jumonji N domain of KDM4D inhibited PARsylation. PARG becomes dispensable for ligand-dependent transcription when either a PARP inhibitor or a non-PARsylable KDM4D/JMJD2D mutant is used. Our results define PARG as a coactivator regulating chromatin remodeling during RA-dependent gene expression.

New readers and interpretations of poly(ADP-ribosyl)ation.

Poly(ADP-ribosyl)ation (PARylation), a protein post-translational modification that was originally connected to the DNA damage response, is now known to engage in a continuously increasing number of biological processes. Despite extensive research and ceaseless, important findings about its role and mode of action, poly(ADP-ribose) remains an enigma regarding its structural complexity and diversity. The recent identification and structural characterization of four different poly(ADP-ribose) binding motifs represents a quantum leap in the comprehension of how this molecule can be decoded. Moreover, the recent discovery of a direct connection between PARylation and poly-ubiquitylation in targeting proteins for degradation by the proteasome has paved the way for a new interpretation of this protein modification. These two novel aspects, poly(ADP-ribose) recognition and readout by the ubiquitylation/proteasome system are developed here.

Purification of recombinant poly(ADP-ribose) polymerases.

The purification of Poly(ADP-ribose) polymerases from overexpressing cells (Sf9 insect cells, Escherichia coli) has been updated to a fast and reproducible three chromatographic steps protocol. After cell lysis, proteins from the crude extract are separated on a Heparine Sepharose™ column. The PARP-containing fractions are then affinity purified on a 3-aminobenzamide Sepharose™ chromatographic step. The last contaminants and the 3-methoxybenzamide used to elute the PARP from the previous affinity column are removed on the high-performance strong cations exchanger Source™ 15S matrix. The columns connected to an ÄKTA™ purifier system allow the purification of PARPs in 3 days with a high-yield recovery. As described in the protocol, more than 11 mg of pure and highly active mouse PARP-2 can be obtained from 1 L of Sf9 insect cell culture.

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.

Radiation-induced mitotic catastrophe in PARG-deficient cells.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in the regulation of chromatin structure, DNA metabolism, cell division and cell death. Through the hydrolysis of poly(ADP-ribose) (PAR), Poly(ADP-ribose) glycohydrolase (PARG) has a crucial role in the control of life-and-death balance following DNA insult. Comprehension of PARG function has been hindered by the existence of many PARG isoforms encoded by a single gene and displaying various subcellular localizations. To gain insight into the function of PARG in response to irradiation, we constitutively and stably knocked down expression of PARG isoforms in HeLa cells. PARG depletion leading to PAR accumulation was not deleterious to undamaged cells and was in fact rather beneficial, because it protected cells from spontaneous single-strand breaks and telomeric abnormalities. By contrast, PARG-deficient cells showed increased radiosensitivity, caused by defects in the repair of single- and double-strand breaks and in mitotic spindle checkpoint, leading to alteration of progression of mitosis. Irradiated PARG-deficient cells displayed centrosome amplification leading to mitotic supernumerary spindle poles, and accumulated aberrant mitotic figures, which induced either polyploidy or cell death by mitotic catastrophe. Our results suggest that PARG could be a novel potential therapeutic target for radiotherapy.

Detection of the nuclear poly(ADP-ribose)-metabolizing enzymes and activities in response to DNA damage.

Poly(ADP-ribosyl)ation is a posttranslational modification of proteins in higher eukaryotes mediated by poly(ADP-ribose) polymerases (PARPs) that is involved in many physiological processes such as DNA repair, transcription, cell division, and cell death. Biochemical studies together with PARP-1- or PARP-2-deficient cellular and animal models have revealed the redundant but also complementary functions of the two enzymes in the surveillance and maintenance of genome integrity. Poly(ADP-ribose) is degraded by the endo- and exo-glycosidase activities of poly(ADP-ribose) glycohydrolase (PARG). In this chapter, biochemical and immunofluorescence methods are described for detecting and assaying PARPs and PARG.

Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells.

Genome integrity is constantly threatened by DNA lesions arising from numerous exogenous and endogenous sources. Survival depends on immediate recognition of these lesions and rapid recruitment of repair factors. Using laser microirradiation and live cell microscopy we found that the DNA-damage dependent poly(ADP-ribose) polymerases (PARP) PARP-1 and PARP-2 are recruited to DNA damage sites, however, with different kinetics and roles. With specific PARP inhibitors and mutations, we could show that the initial recruitment of PARP-1 is mediated by the DNA-binding domain. PARP-1 activation and localized poly(ADP-ribose) synthesis then generates binding sites for a second wave of PARP-1 recruitment and for the rapid accumulation of the loading platform XRCC1 at repair sites. Further PARP-1 poly(ADP-ribosyl)ation eventually initiates the release of PARP-1. We conclude that feedback regulated recruitment of PARP-1 and concomitant local poly(ADP-ribosyl)ation at DNA lesions amplifies a signal for rapid recruitment of repair factors enabling efficient restoration of genome integrity.