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.     

Down-regulation of DNA repair synthesis at DNA single-strand interruptions in poly(ADP-ribose) polymerase-1 deficient murine cell extracts

The functional involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in the repair of DNA single- and double-strand breaks, DNA base damage, and related repair substrate intermediates remains unclear. Using an in vitro DNA repair assay and cell extracts derived from PARP-1 deficient or wild-type murine embryonic fibroblasts, we investigated the DNA synthesis and ligation steps associated with the rejoining of DNA single-strand interruptions containing 3'-OH, and either 5'-OH or 5'-P termini. Complete repair leading to DNA rejoining was similar between PARP-1 deficient cells and wild-type controls and poly(ADP-ribose) synthesis was, as expected, greatly reduced in PARP-1 deficient cell extracts. The incorporation of [32P]dCMP into repaired DNA at the site of a lesion was reduced two-three-fold in PARP-1 deficient cell extracts, demonstrating a decrease in repair patch size. Addition of purified PARP-1 to levels approximating those present in wild-type extracts did not stimulate DNA repair synthesis. We conclude that PARP-1 is not required for the efficient processing and rejoining of single-strand interruptions with defined 3'-OH and 5'-OH or 5'-P termini. Decreased DNA repair synthesis observed in PARP-1 deficient cell extracts is associated with reduced cellular expression of several factors required for long-patch base excision repair (BER), including FEN-1 and DNA ligase I.     

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.     

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(ADP-ribose) polymerase-1 modulates DNA repair capacity and prevents formation of DNA double strand breaks

Although poly(ADP-ribose) polymerase-1 (PARP-1) has no enzymatic activity involved in DNA damage processing by the base excision repair (BER) pathway, PARP-1 deficient cells are genetically unstable and sensitive to DNA-damaging agents. To explain this paradox, we investigated the impact of PARP-1 on BER in mammalian cells. We reduced cellular PARP-1 protein levels using siRNA, then introduced DNA damage by hydrogen peroxide treatment and examined the repair response. We find that PARP-1 is not involved in recruitment of the major BER proteins to sites of DNA damage. However, we find that PARP-1 protects excessive DNA single strand breaks (SSBs) from converting into DNA double strand breaks (DSBs) thus preserving them for subsequent repair by BER enzymes. This suggests that PARP-1 plays an important role in BER by extending the ability of BER enzymes to process DNA single strand breaks arising directly after mutagen stress or during processing of DNA lesions following extensive DNA damage.     

Poly(ADP-ribose) polymerase-1: what have we learned from the deficient mouse model?

Poly (ADP-ribose) polymerase (113 kDa; PARP-1) is a constitutive factor of the DNA damage surveillance network developed by the eukaryotic cell to cope with the numerous environmental and endogenous genotoxic agents. This enzyme recognizes and is activated by DNA strand breaks. This original property plays an essential role in the protection and processing of the DNA ends as they arise in DNA damage that triggers the base excision repair (BER) pathway. The generation, by homologous recombination, of three independent deficient mouse models have confirmed the caretaker function of PARP-1 in mammalian cells under genotoxic stress. Unexpectedly, the knockout strategy has revealed the instrumental role of PARP-1 in cell death after ischemia-reperfusion injury and in various inflammation process. Moreover, the residual PARP activity found in PARP-1 deficient cells has been recently attributed to a novel DNA damage-dependent poly ADP-ribose polymerase (62 kDa; PARP-2), another member of the expanding PARP family that, on the whole, appears to be involved in the genome protection. The present review summarizes the recent data obtained with the three PARP knockout mice in comparison with the chemical inhibitor approach.     

Calcium-dependent modulation of poly(ADP-ribose) polymerase-1 alters cellular metabolism and DNA repair

After genotoxic stress poly(ADP-ribose) polymerase-1 (PARP-1) can be hyperactivated, causing (ADP-ribosyl)ation of nuclear proteins (including itself), resulting in NAD(+) and ATP depletion and cell death. Mechanisms of PARP-1-mediated cell death and downstream proteolysis remain enigmatic. beta-lapachone (beta-lap) is the first chemotherapeutic agent to elicit a Ca(2+)-mediated cell death by PARP-1 hyperactivation at clinically relevant doses in cancer cells expressing elevated NAD(P)H:quinone oxidoreductase 1 (NQO1) levels. Beta-lap induces the generation of NQO1-dependent reactive oxygen species (ROS), DNA breaks, and triggers Ca(2+)-dependent gamma-H2AX formation and PARP-1 hyperactivation. Subsequent NAD(+) and ATP losses suppress DNA repair and cause cell death. Reduction of PARP-1 activity or Ca(2+) chelation protects cells. Interestingly, Ca(2+) chelation abrogates hydrogen peroxide (H(2)O(2)), but not N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PARP-1 hyperactivation and cell death. Thus, Ca(2+) appears to be an important co-factor in PARP-1 hyperactivation after ROS-induced DNA damage, which alters cellular metabolism and DNA repair.     

Poly(ADP-ribose) polymerase-1 as a regulator of protein-nucleic acid interactions in the processes responding to genotoxic action

Poly(ADP-ribose) polymerase-1 (PARP-1), nuclear protein of higher eukaryotes, specifically detects strand breaks in DNA. When bound to DNA strand breaks, PARP-1 is activated and catalyzes synthesis of poly(ADP-ribose) covalently attached to the row of nuclear proteins, with the main acceptor being PARP-1 itself. This protein participates in a majority of DNA dependent processes: repair, recombination; replication: cell death: apoptosis and necrosis. Poly(ADP-ribosyl)ation of proteins is considered as mechanism, which signals about DNA damage and modulate protein functioning in response to genotoxic impact. The main emphasis is made on the roles of PARP-1 and poly(ADP-ribosyl)ation in base excision repair (BER), the process, which provides repair of DNA breaks. The main proposed functions of PARP-1 in this process are: factor initiating assemblage of protein complex of BER; temporary protection of DNA ends; modulation of chromatin structure via poly(ADP-ribosyl)ation of histones; signaling function in detection of the levels of DNA damage in cell.     

NAD+ repletion prevents PARP-1-induced glycolytic blockade and cell death in cultured mouse astrocytes

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that is involved in DNA repair and activated by DNA damage. When activated, PARP-1 consumes NAD(+) to form ADP-ribose polymers on acceptor proteins. Extensive activation of PARP-1 leads to glycolytic blockade, energy failure, and cell death. These events have been postulated to result from NAD(+) depletion. Here, we used primary astrocyte cultures to directly test this proposal, utilizing the endogenous expression of connexin-43 hemichannels by astrocytes to manipulate intracellular NAD(+) concentrations. Activation of PARP-1 with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) produced NAD(+) depletion, glycolytic blockade, and cell death. Cultures incubated in high (10mM) extracellular concentrations of NAD(+) after MNNG exposure showed normalization of intracellular NAD(+) concentrations. Repletion of intracellular NAD(+) in this manner completely restored glycolytic capacity and prevented cell death. These results suggest that NAD(+) depletion is the cause of glycolytic failure after PARP-1 activation.     

Poly(ADP-ribosyl)ation accelerates DNA repair in a pathway dependent on Cockayne syndrome B protein

Activation of poly(ADP-ribose)polymerases 1 and 2 (PARP-1 and PARP-2) is one of the earliest responses of mammalian cells to DNA damage by numerous genotoxic agents. We have analysed the influence of PARP inhibition, either achieved by over-expression of the DNA binding domain of PARP-1 or by treatment with 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone, on the repair of single-strand breaks (SSB), pyrimidine dimers and oxidative base modifications sensitive to Fpg protein (mostly 8-hydroxyguanine) in mammalian cells at very low, non-cytotoxic levels of DNA damage. The data show that the repair rates of all three types of DNA damage are significantly lower in PARP-inhibited cells. Importantly, the retardation of the repair of base modifications is not associated with accumulation of intermediates such as SSB or abasic sites. Moreover, the influence of the PARP inhibition is not observed in cells deficient in Cockayne syndrome B protein (Csb). The results indicate that PARP activation and Csb are both involved in a novel mechanism that accelerates the global repair of various types of DNA modifications.     

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 Inhibits Strand-Displacement Synthesis of DNA Catalyzed by DNA Polymerase β

Poly(ADP-ribose) polymerase-1 (PARP-1), a eucaryotic nuclear DNA-binding protein that is activated by breaks in DNA chains, may be involved in the base excision repair (BER) because DNAs containing single-stranded gaps and breaks are intermediates of BER. The effect of PARP-1 on the DNA synthesis catalyzed in vitro by DNA polymerase beta (pol beta) was studied using analogs of DNA substrates produced during BER and imitating intermediates of the short patch and long patch subpathways of BER. Oligonucleotide duplexes of 34 bp that contained a mononucleotide gap or a single-strand break with tetrahydrofuran phosphate or phosphate at the 5;-end of the downstream oligonucleotide were taken as DNA substrates. The efficiency of DNA synthesis was determined at various ratios of pol beta and PARP-1. The efficiency of gap filling was decreased in the presence of PARP-1, but strand-displacement DNA synthesis was inhibited significantly stronger, which seemed to be due to competition between PARP-1 and pol beta for DNA. In the presence of NAD+ and single-strand breaks in DNA, PARP-1 catalyzes the synthesis of poly(ADP-ribose) covalently attached to the enzyme, and this automodification is thought to provide for dissociation of PARP-1 from DNA. The effect of PARP-1 automodification on inhibition of DNA synthesis was studied, and efficiency of mononucleotide gap filling was shown to be restored, but strand-displacement synthesis did not revert to the level observed in the absence of PARP-1. PARP-1 is suggested to regulate the interaction between pol beta and DNA, in particular, via its own automodification.     

Hyperactivation of PARP Triggers Nonhomologous End-Joining in Repair-Deficient Mouse Fibroblasts

Regulation of poly(ADP-ribose) (PAR) synthesis and turnover is critical to determining cell fate after genotoxic stress. Hyperactivation of PAR synthesis by poly(ADP-ribose) polymerase-1 (PARP-1) occurs when cells deficient in DNA repair are exposed to genotoxic agents; however, the function of this hyperactivation has not been adequately explained. Here, we examine PAR synthesis in mouse fibroblasts deficient in the base excision repair enzyme DNA polymerase β (pol β). The extent and duration of PARP-1 activation was measured after exposure to either the DNA alkylating agent, methyl methanesulfonate (MMS), or to low energy laser-induced DNA damage. There was strong DNA damage-induced hyperactivation of PARP-1 in pol β nullcells, but not in wild-type cells. In the case of MMS treatment, PAR synthesis did not lead to cell death in the pol β null cells, but instead resulted in increased PARylation of the nonhomologous end-joining (NHEJ) protein Ku70 and increased association of Ku70 with PARP-1. Inhibition of the NHEJ factor DNA-PK, under conditions of MMS-induced PARP-1 hyperactivation, enhanced necrotic cell death. These data suggest that PARP-1 hyperactivation is a protective mechanism triggering the classical-NHEJ DNA repair pathway when the primary alkylated base damage repair pathway is compromised.     

The role of poly(ADP-ribose) in the DNA damage signaling network.

DNA damage signaling is crucial for the maintenance of genome integrity. In higher eukaryotes a NAD+-dependent signal transduction mechanism has evolved to protect cells against the genome destabilizing effects of DNA strand breaks. The mechanism involves 2 nuclear enzymes that sense DNA strand breaks, poly(ADP-ribose) polymerase-1 and -2 (PARP-1 and PARP-2). When activated by DNA breaks, these PARPs use NAD+ to catalyze their automodification with negatively charged, long and branched ADP-ribose polymers. Through recruitment of specific proteins at the site of damage and regulation of their activities, these polymers may either directly participate in the repair process or coordinate repair through chromatin unfolding, cell cycle progression, and cell survival-cell death pathways. A number of proteins, including histones, DNA topoisomerases, DNA methyltransferase-1 as well as DNA damage repair and checkpoint proteins (p23, p21, DNA-PK, NF-kB, XRCC1, and others) can be targeted in this manner; the interaction involves a specific poly(ADP-ribose)-binding sequence motif of 20-26 amino acids in the target domains.     

Poly(ADP-ribose) polymerase-1 protects excessive DNA strand breaks from deterioration during repair in human cell extracts

Base excision repair (BER), a major pathway for the removal of simple lesions in DNA, requires the co-ordinated action of several repair and ancillary proteins, the impairment of which can lead to genetic instability. We here address the role of poly(ADP-ribose) polymerase-1 (PARP-1) in BER. Using an in vitro cross-linking assay, we reveal that PARP-1 is always involved in repair of a uracil-containing oligonucleotide and that it binds to the damaged DNA during the early stages of repair. Inhibition of PARP-1 poly(ADP-ribosyl)ation by 3-aminobenzamide blocks dissociation of PARP-1 from damaged DNA and prevents further repair. We find that excessive poly(ADP-ribosyl)ation occurs when repair intermediates containing single-strand breaks are in excess of the repair capacity of the cell extract, suggesting that repeated binding of PARP-1 to the nicked DNA occurs. We also find increased sensitivity of repair intermediates to nuclease cleavage in PARP-deficient mouse fibroblasts and after depletion of PARP-1 from HeLa whole cell extracts. Our data support the model in which PARP-1 binding to DNA single-strand breaks or repair intermediates plays a protective role when repair is limited.     

PARP-1 enhances the mismatch-dependence of 5'-directed excision in human mismatch repair in vitro

End-directed mismatch-provoked excision has been reconstituted in several purified systems. While 3'-directed excision displays a mismatch dependence similar to that observed in nuclear extracts (≈20-fold), the mismatch dependence of 5'-directed excision is only 3-4-fold, significantly less than that in extracts (8-10-fold). Utilizing a fractionation-based approach, we have isolated a single polypeptide that enhances mismatch dependence of reconstituted 5'-directed excision and have shown it to be identical to poly[ADP-ribose] polymerase 1 (PARP-1). Titration of reconstituted excision reactions or PARP-1-depleted HeLa nuclear extract with purified PARP-1 showed that the protein specifically enhances mismatch dependence of 5'-directed excision. Analysis of a set of PARP-1 mutants revealed that the DNA binding domain and BRCT fold contribute to the regulation of excision specificity. Involvement of the catalytic domain is restricted to its ability to poly(ADP-ribosyl)ate PARP-1 in the presence of NAD(+), likely through interference with DNA binding. Analysis of protein-protein interactions demonstrated that PARP-1 interacts with mismatch repair proteins MutSα, exonuclease 1, replication protein A (RPA), and as previously shown by others, replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) as well. The BRCT fold plays an important role in the interaction of PARP-1 with the former three proteins.     

AP endonuclease and poly(ADP-ribose) polymerase-1 interact with the same base excision repair intermediate

Base excision repair (BER) is a defense system that protects cells from deleterious effects secondary to modified or missing DNA bases. BER is known to involve apurinic/apyrimidinic endonuclease (APE) and DNA polymerase ss (ss-pol) among other enzymes, and recent studies have suggested that poly(ADP-ribose) polymerase-1 (PARP-1) also plays a role by virtue of its binding to BER intermediates. The main role of APE is cleavage of the DNA backbone at abasic sites, and the enzyme also can catalyze 3'- to 5'-exonuclease activity at the cleaved abasic site. Photocross-linking studies with mouse embryonic fibroblast (MEF) cell extracts described here indicated that APE and PARP-1 interact with the same APE-cleaved abasic site BER intermediate. The model BER intermediate used includes a synthetic abasic site sugar, i.e. tetrahydrofuran (THF), in place of the natural deoxyribose. APE cross-linked efficiently with this intermediate, but not with a molecule lacking the 5'-THF phosphate group, and the same property was demonstrated for PARP-1. The addition of purified APE to the MEF extract reduced the amount of PARP-1 cross-linked to the BER intermediate, suggesting that APE can compete with PARP-1. APE and PARP-1 were antagonists of each other in in vitro BER related reactions on this model BER intermediate. These results suggest that PARP-1 and APE can interact with the same BER intermediate and that competition between these two proteins may influence their respective BER related functions.