Base excision repair is impaired in mammalian cells lacking Poly(ADP-ribose) polymerase-1

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) in the skin and in melanomas

Cutaneous melanoma (CM) and uveal melanoma (UM) represent the most aggressive pigment cell tumor types. Our investigation examined the signaling molecule poly(ADP-ribose) (PAR) in CM and UM. We have demonstrated PAR in keratinocytes, sebocytes, hair follicles, endothelial cells and in subcutaneous adipocytes in the normal skin indicating that PAR may regulate physiological functions in these cell types. Furthermore, CM cells were PAR positive and tumor invasion level/thickness of CM correlated with the PAR content of the cell nuclei, with higher Clark and Breslow indices and AJCC scores associating with higher PAR content. This correlation was especially marked in the samples of female patients. In UM tumors (n=12) a slight overall and strong perivascular PAR staining was observed with considerable individual variations. In view of recent successful clinical trials with PARP inhibitors as adjuvant chemotherapeutic agents, our results suggest that melanomas may display differential sensitivity towards this novel therapeutic modality which should be considered for the selection of patients.     

Involvement of poly(ADP-ribose) polymerase in base excision repair

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.     

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.     

Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate

Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.     

Poly(ADP-ribose) polymerase in base excision repair: always engaged,but not essential for DNA damage processing

Poly(ADP-ribose) polymerase (PARP-1) is an abundant nuclear protein with a high affinity for single- and double-strand DNA breaks. Its binding to strand breaks promotes catalysis of the covalent modification of nuclear proteins with poly(ADP-ribose) synthesised from NAD(+). PARP-1-knockout cells are extremely sensitive to alkylating agents, suggesting the involvement of PARP-1 in base excision repair; however, its role remains unclear. We investigated the dependence of base excision repair pathways on PARP-1 and NAD(+) using whole cell extracts derived from normal and PARP-1 deficient mouse cells and DNA substrates containing abasic sites. In normal extracts the rate of repair was highly dependent on NAD(+). We found that in the absence of NAD(+) repair was slowed down 4-6-fold after incision of the abasic site. We also established that in extracts from PARP-1 deficient mouse cells, repair of both regular and reduced abasic sites was increased with respect to normal extracts and was NAD(+)-independent, suggesting that in both short- and long-patch BER PARP-1 slows down, rather than stimulates, the repair reaction. Our data support the proposal that PARP-1 does not play a major role in catalysis of DNA damage processing via either base excision repair pathway.     

Participation of Poly(ADP-ribose)-polymerase of nuclear matrix in DNA repair

The poly(ADP-ribose)-polymerase activity of brain and liver cell nuclei is changed during X-irradiation of rats. In the nuclear matrix, poly(ADP-ribose)-polymerase activity increases at a low dose of irradiation (1.7 Gy) and decreases at a high dose (6.7 Gy). A significant part of the activity of nuclear NMN-adenylyltransferase, a key enzyme for biosynthesis of NAD (the substrate of poly(ADP-ribose)-polymerase), has been found in the nuclear matrix. An interrelation between ADP-ribosylation taking place on the matrix level and eukaryotic cell DNA repair is suggested.     

Base excision repair is efficient in cells lacking poly(ADP-ribose) polymerase 1

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is activated by binding to DNA breaks induced by ionizing radiation or through repair of altered bases in DNA by base excision repair. Mice lacking PARP-1 and, in certain cases, the cells derived from these mice exhibit hypersensitivity to ionizing radiation and alkylating agents. In this study we investigated base excision repair in cells lacking PARP-1 in order to elucidate whether their augmented sensitivity to DNA damaging agents is due to an impairment of the base excision repair pathway. Extracts prepared from wild-type cells or cells lacking PARP-1 were similar in their ability to repair plasmid DNA damaged by either X-rays (single-strand DNA breaks) or by N-methyl-N′-nitro-N-nitrosoguanidine (methylated bases). In addition, we demonstrated in vivo that PARP-1-deficient cells treated with N-methyl-N′-nitro-N-nitrosoguanidine repaired their genomic DNA as efficiently as wild-type cells. Therefore, we conclude that cells lacking PARP-1 have a normal capacity to repair single-strand DNA breaks inflicted by X-irradiation or breaks formed during the repair of modified bases. We propose that the hypersensitivity of PARP-1 null mutant cells to γ-irradiation and alkylating agents is not directly due to a defect in DNA repair itself, but rather results from greatly reduced poly(ADP-ribose) formation during base excision repair in these cells.     

Investigations on dark repair in various light sensitive inherited disorders

Summary Defective dark repair is confirmed in 3 cases of Xeroderma pigmentosum while in one case of Bloom-syndrom and in 4 other light sensitive inherited disorders the mentioned enzyme system seems to be functional.

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Zusammenfassung Ein defekter dark repair-Mechanismus wurde bei 3 Fällen von Xeroderma pigmentosum (X.p.) bestätigt, während bei einem Fall von Bloom-Syndrom und 4 anderen lichtabhängigen Erbleiden der Haut normal funktionierende Repair-Enzyme gefunden wurden.

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This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Jn 80/1).     

多ADP-核糖聚合酶家族

分离鉴定多功能的核基质蛋白及核基质结合蛋白是目前核基质研究的一个重要领域。通过与转录因子、核基质结合元件以及DNA间相互作用,核基质结合蛋白在DNA复制、转录、加工修饰等细胞内事件中起着支持和调节的作用。多ADP-核糖聚合酶[poly(ADP—ribose)polymerase,PARP]是一种高度保守的核基质结合蛋白,在多种活动例如基因组损伤修复、细胞凋亡、信号转导、基因表达调控中都发挥着调节的功能。PARP的潜在生物学功能已越来越引起国内外研究人员的关注。     

Poly(ADP-ribose) polymerase-2 (PARP-2) is required for efficient base excision DNA repair in association with PARP-1 and XRCC1

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(etheno ADP-ribose) blocks poly(ADP-ribose) glycohydrolase activity

Poly(ADP-ribose) is a biopolymer synthesized by poly(ADP-ribose) polymerases. Recent findings suggest the possibility for modulation of cellular functions including cell death and mitosis by poly(ADP-ribose). Derivatization of poly(ADP-ribose) may be useful for investigating the effects of poly(ADP-ribose) on various cellular processes. We prepared poly(etheno ADP-ribose) (poly(epsilonADP-ribose)) by converting the adenine moiety of poly(ADP-ribose) to 1-N(6)-etheno adenine residues. Poly(epsilonADP-ribose) is shown to be highly resistant to digestion by poly(ADP-ribose) glycohydrolase (Parg). On the other hand, poly(epsilonADP-ribose) could be readily digested by phosphodiesterase. Furthermore, poly(epsilonADP-ribose) inhibited Parg activity to hydrolyse ribose-ribose bonds of poly(ADP-ribose). This study suggests the possibility that poly(epsilonADP-ribose) might be a useful tool for studying the poly(ADP-ribose) dynamics and function of Parg. This study also implies that modification of the adenine moiety of poly(ADP-ribose) abrogates the susceptibility to digestion by Parg.     

DNA polymerase beta -mediated long patch base excision repair. Poly(ADP-ribose)polymerase-1 stimulates strand displacement DNA synthesis.

Recently, photoaffinity labeling experiments with mouse cell extracts suggested that PARP-1 functions as a surveillance protein for a stalled BER intermediate. To further understand the role of PARP-1 in BER, we examined the DNA synthesis and flap excision steps in long patch BER using a reconstituted system containing a 34-base pair BER substrate and five purified human enzymes: uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta, flap endonuclease-1 (FEN-1), and PARP-1. PARP-1 stimulates strand displacement DNA synthesis by DNA polymerase beta in this system; this stimulation is dependent on the presence of FEN-1. PARP-1 and FEN-1, therefore, cooperate to activate long patch BER. The results are discussed in the context of a model for BER sub-pathway choice, illustrating a dual role for PARP-1 as a surveillance protein for a stalled BER intermediate and an activating factor for long patch BER DNA synthesis.     

Poly(ADP-ribose): PARadigms and PARadoxes

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational protein modification (PTM) catalyzed by members of the poly(ADP-ribose) polymerase (PARP) enzyme family. PARPs use NAD⁺ as substrate and upon cleaving off nicotinamide they transfer the ADP-ribosyl moiety covalently to suitable acceptor proteins and elongate the chain by adding further ADP-ribose units to create a branched polymer, termed poly(ADP-ribose) (PAR), which is rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). In recent years several key discoveries changed the way we look at the biological roles and mode of operation of PARylation. These paradigm shifts include but are not limited to (1) a single PARP enzyme expanding to a PARP family; (2) DNA-break dependent activation extended to several other DNA dependent and independent PARP-activation mechanisms; (3) one molecular mechanism (covalent PARylation of target proteins) underlying the biological effect of PARPs is now complemented by several other mechanisms such as protein–protein interactions, PAR signaling, modulation of NAD⁺ pools and (4) one principal biological role in DNA damage sensing expanded to numerous, diverse biological functions identifying PARP-1 as a real moonlighting protein. Here we review the most important paradigm shifts in PARylation research and also highlight some of the many controversial issues (or paradoxes) of the field such as (1) the mostly synergistic and not antagonistic biological effects of PARP-1 and PARG; (2) mitochondrial PARylation and PAR decomposition, (3) the cross-talk between PARylation and signaling pathways (protein kinases, phosphatases, calcium) and the (4) divergent roles of PARP/PARylation in longevity and in age-related diseases.     

Poly(ADP-ribose) signaling in cell death

Poly(ADP-ribosyl)ation (PARylation) is a reversible protein modification carried out by the concerted actions of poly(ADP-ribose) polymerase (PARP) enzymes and poly(ADP-ribose) (PAR) decomposing enzymes such as PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Reversible PARylation is a pleiotropic regulator of various cellular functions but uncontrolled PARP activation may also lead to cell death. The cellular demise pathway mediated by PARylation in oxidatively stressed cells has been described almost thirty years ago. However, the underlying molecular mechanisms have only begun to emerge relatively recently. PARylation has been implicated in necroptosis, autophagic cell death but its role in extrinsic and intrinsic apoptosis appears to be less predominant and depends largely on the cellular model used. Currently, three major pathways have been made responsible for PARP-mediated necroptotic cell death: (1) compromised cellular energetics mainly due to depletion of NAD, the substrate of PARPs; (2) PAR mediated translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus (parthanatos) and (3) a mostly elusive crosstalk between PARylation and cell death/survival kinases and phosphatases. Here we review how these PARP-mediated necroptotic pathways are intertwined, how PARylation may contribute to extrinsic and intrinsic apoptosis and discuss recent developments on the role of PARylation in autophagy and autophagic cell death.     

Poly(ADP-ribose) catabolism in mammalian cells

Poly(ADP-ribose) catabolism is a complex situation involving many proteins and DNA. We have developed anin vitro turnover system where poly(ADP-ribose) metabolism is monitored in presence of different relative amounts of two principal enzymes poly(ADP-ribose) transferase and poly(ADP-ribose) glycohydrolase along with other proteins and DNA. Our current results reviewed here show that the quality of polymer, i.e. chain length and complexity, as well as preference for the nuclear substrate varies depending upon the availability of poly(ADP-ribose) glycohydrolase. These results are interpreted in the light of the recent data implicating poly(ADP-ribose) metabolism in DNA-repair. (Mol Cell Biochem 138: 45–52 1994)     

Poly(ADP-ribose) synthesis and cell division

A clone of HeLa cells has been selected in the presence of 5-methylnicotinamide. In the presence of 10 mM 5-methylnicotinamide the resistant cells grow at 70% of the rate of the same cell culture without 5-methylnicotinamide. 10 mM 5-methylnicotinamide completely inhibits the growth of normal HeLa cells. Both cell types in the absence of 5-methylnicotinamide have the same generation time. Poly (adenosine diphosphate-ribose) synthesis is less sensitive to 5-methylnicotinamide in nuclei isolated from the resistant cells than in nuclei from sensitive cells.