Cellular responses and repair of single-strand breaks introduced by UV damage endonuclease in mammalian cells

Although single-strand breaks (SSBs) occur frequently, the cellular responses and repair of SSB are not well understood. To address this, we established mammalian cell lines expressing Neurospora crassa UV damage endonuclease (UVDE), which introduces a SSB with a 3'-OH immediately 5' to UV-induced cyclobutane pyrimidine dimers or 6-4 photoproducts and initiates an alternative excision repair process. Xeroderma pigmentosum group A cells expressing UVDE show UV resistance of almost the wild-type level. In these cells SSBs are produced upon UV irradiation and then efficiently repaired. The repair patch size is about seven nucleotides, and repair synthesis is decreased to 30% by aphidicolin, suggesting the involvement of a DNA polymerase delta/epsilon-dependent long-patch repair. Immediately after UV irradiation, cellular proteins are poly(ADP-ribosyl)ated. The UV resistance of the cells is decreased in the presence of 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase. Expression of UVDE in XRCC1-defective EM9, a Chinese hamster ovary cell line, greatly sensitizes the host cells to UV, and addition of 3-aminobenzamide results in almost no further sensitization of the cells to UV. Thus, we show that XRCC1 and PARP are involved in the same pathway for the repair of SSBs.     

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.     

XRCC1 interacts with the p58 subunit of DNA Pol α-primase and may coordinate DNA repair and replication during S phase

Repair of single-stranded DNA breaks before DNA replication is critical in maintaining genomic stability; however, how cells deal with these lesions during S phase is not clear. Using combined approaches of proteomics and in vitro and in vivo protein–protein interaction, we identified the p58 subunit of DNA Pol α-primase as a new binding partner of XRCC1, a key protein of the single strand break repair (SSBR) complex. In vitro experiments reveal that the binding of poly(ADP-ribose) to p58 inhibits primase activity by competition with its DNA binding property. Overexpression of the XRCC1-BRCT1 domain in HeLa cells induces poly(ADP-ribose) synthesis, PARP-1 and XRCC1-BRCT1 poly(ADP-ribosyl)ation and a strong S phase delay in the presence of DNA damage. Addition of recombinant XRCC1-BRCT1 to Xenopus egg extracts slows down DNA synthesis and inhibits the binding of PCNA, but not MCM2 to alkylated chromatin, thus indicating interference with the assembly of functional replication forks. Altogether these results suggest a critical role for XRCC1 in connecting the SSBR machinery with the replication fork to halt DNA synthesis in response to DNA damage.     

APLF (C2orf13) is a novel component of poly(ADP-ribose) signaling in mammalian cells

APLF is a novel protein of unknown function that accumulates at sites of chromosomal DNA strand breakage via forkhead-associated (FHA) domain-mediated interactions with XRCC1 and XRCC4. APLF can also accumulate at sites of chromosomal DNA strand breaks independently of the FHA domain via an unidentified mechanism that requires a highly conserved C-terminal tandem zinc finger domain. Here, we show that the zinc finger domain binds tightly to poly(ADP-ribose), a polymeric posttranslational modification synthesized transiently at sites of chromosomal damage to accelerate DNA strand break repair reactions. Protein poly(ADP-ribosyl)ation is tightly regulated and defects in either its synthesis or degradation slow global rates of chromosomal single-strand break repair. Interestingly, APLF negatively affects poly(ADP-ribosyl)ation in vitro, and this activity is dependent on its capacity to bind the polymer. In addition, transient overexpression in human A549 cells of full-length APLF or a C-terminal fragment encoding the tandem zinc finger domain greatly suppresses the appearance of poly(ADP-ribose), in a zinc finger-dependent manner. We conclude that APLF can accumulate at sites of chromosomal damage via zinc finger-mediated binding to poly(ADP-ribose) and is a novel component of poly(ADP-ribose) signaling in mammalian cells.     

Poly(ADP-ribose) Polymerase 1 Modulates Interaction of the Nucleotide Excision Repair Factor XPC-RAD23B with DNA via Poly(ADP-ribosyl)ation

Poly(ADP-ribosyl)ation is a reversible post-translational modification that plays an essential role in many cellular processes, including regulation of DNA repair. Cellular DNA damage response by the synthesis of poly(ADP-ribose) (PAR) is mediated mainly by poly(ADP-ribose) polymerase 1 (PARP1). The XPC-RAD23B complex is one of the key factors of nucleotide excision repair participating in the primary DNA damage recognition. By using several biochemical approaches, we have analyzed the influence of PARP1 and PAR synthesis on the interaction of XPC-RAD23B with damaged DNA. Free PAR binds to XPC-RAD23B with an affinity that depends on the length of the poly(ADP-ribose) strand and competes with DNA for protein binding. Using ³²P-labeled NAD⁺ and immunoblotting, we also demonstrate that both subunits of the XPC-RAD23B are poly(ADP-ribosyl)ated by PARP1. The efficiency of XPC-RAD23B PARylation depends on DNA structure and increases after UV irradiation of DNA. Therefore, our study clearly shows that XPC-RAD23B is a target of poly(ADP-ribosyl)ation catalyzed by PARP1, which can be regarded as a universal regulator of DNA repair processes.     

Ataxia telangiectasia mutated (ATM) signaling network is modulated by a novel poly(ADP-ribose)-dependent pathway in the early response to DNA-damaging agents

Poly(ADP-ribosyl)ation is a post-translational modification that is instantly stimulated by DNA strand breaks creating a unique signal for the modulation of protein functions in DNA repair and cell cycle checkpoint pathways. Here we report that lack of poly(ADP-ribose) synthesis leads to a compromised response to DNA damage. Deficiency in poly(ADP-ribosyl)ation metabolism induces profound cellular sensitivity to DNA-damaging agents, particularly in cells deficient for the protein kinase ataxia telangiectasia mutated (ATM). At the biochemical level, we examined the significance of poly(ADP-ribose) synthesis on the regulation of early DNA damage-induced signaling cascade initiated by ATM. Using potent PARP inhibitors and PARP-1 knock-out cells, we demonstrate a functional interplay between ATM and poly(ADP-ribose) that is important for the phosphorylation of p53, SMC1, and H2AX. For the first time, we demonstrate a functional and physical interaction between the major DSB signaling kinase, ATM and poly(ADP-ribosyl)ation by PARP-1, a key enzyme of chromatin remodeling. This study suggests that poly(ADP-ribose) might serve as a DNA damage sensory molecule that is critical for early DNA damage signaling.     

DNA strand break metabolism in human lymphocytes: a reevaluation

Recent findings concerning the presumed existence of single-strand breaks (SSB) in quiescent human peripheral blood lymphocytes (PBL) are discussed in relation to the role of poly(ADP-ribosyl)ation in DNA strand break metabolism. It is argued that the activation of poly(ADP-ribose)polymerase (ADPRP) by a DNA-damaging agent is not indicative of an obligatory role of poly(ADP-ribosyl)ation in DNA repair. From this it follows that SSB induced by different strand-breaking agents might be removed by either ADPRP-dependent or ADPRP-independent DNA repair pathways.     

XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.     

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.     

The emerging role of poly(ADP-ribose) polymerase-1 in longevity

In the present paper, the involvement of the family of poly(ADP-ribose) polymerases (PARPs), and especially of PARP-1, in mammalian longevity is reviewed. PARPs catalyse poly(ADP-ribosyl)ation, a covalent post-translational protein modification in eukaryotic cells. PARP-1 and PARP-2 are activated by DNA strand breaks, play a role in DNA base-excision repair (BER) and are survival factors for cells exposed to low doses of ionising radiation or alkylating agents. PARP-1 is the main catalyst of poly(ADP-ribosyl)ation in living cells under conditions of DNA breakage, accounting for about 90% of cellular poly(ADP-ribose). DNA-damage-induced poly(ADP-ribosyl)ation also functions as a negative regulator of DNA damage-induced genomic instability. Cellular poly(ADP-ribosyl)ation capacity in permeabilised mononuclear blood cells (MNC) is positively correlated with life span of mammalian species. Furthermore PARP-1 physically interacts with WRN, the protein deficient in Werner syndrome, a human progeroid disorder, and PARP-1 and WRN functionally cooperate in preventing carcinogenesis in vivo. Some of the other members of the PARP family have also been revealed as important regulators of cellular functions relating to ageing/longevity. In particular, tankyrase-1, tankyrase-2, PARP-2 as well as PARP-1 have been found in association with telomeric DNA and are able to poly(ADP-ribosyl)ate the telomere-binding proteins TRF-1 and TRF-2, thus blocking their DNA-binding activity and controlling telomere extension by telomerase.     

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.     

Misregulation of poly(ADP-ribose) polymerase-1 activity and cell type-specific loss of poly(ADP-ribose) synthesis in the cerebellum of aged rats

Protein modification by ADP-ribose polymers is a common regulatory mechanism in eukaryotic cells and is involved in several aspects of brain physiology and physiopathology, including neurotransmission, memory formation, neurotoxicity, ageing and age-associated diseases. Here we show age-related misregulation of poly(ADP-ribose) synthesis in rat cerebellum as revealed by: (i) reduced poly(ADP-ribose) polymerase-1 (PARP-1) activation in response to enzymatic DNA cleavage, (ii) altered protein poly(ADP-ribosyl)ation profiles in isolated nuclei, and (iii) cell type-specific loss of poly(ADP-ribosyl)ation capacity in granule cell layer and Purkinje cells in vivo. In particular, although PARP-1 could be detected in virtually all granule cells, only a fraction of them appeared to be actively engaged in poly(ADP-ribose) synthesis and this fraction was reduced in old rat cerebellum. NAD(+), quantified in tissue homogenates, was essentially the same in the cerebellum of young and old rats suggesting that in vivo factors other than PARP-1 content and/or NAD(+) levels may be responsible for the age-associated lowering of poly(ADP-ribose) synthesis. Moreover, PARP-1 expression was substantially down-regulated in Purkinje cells of senescent rats.     

Potentiation of DNA damage by inhibition of poly(ADP-ribosyl)ation: a test of the hypothesis for random nuclease action.

Poly(ADP-ribosyl)ation is a cellular response to DNA strand breaks by which a large array of proteins becomes covalently modified for a brief period during the lifetime of the DNA breaks. Inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide after many types of DNA damage leads to a marked increase in DNA strand breakage, repair replication, cytogenetic damage, mutagenesis, and cell killing. It has been hypothesized that poly(ADP-ribose) polymerase may modify potentially degradative endogenous nucleases that can reduce cellular viability. Thus, in the presence of DNA strand breakage, the polymer would bind these enzymes to inhibit their activity. When synthesis of the polymerase is inhibited, the enzymes would act randomly to produce nonspecific damage in the DNA. We tested this hypothesis by electroporating restriction enzymes into human cells containing the shuttle vector pHAZE. Restriction enzymes cleave at specific recognition sequences in the lacZ target gene of pHAZE, and mutations result from rejoining errors at the cleavage sites. If the hypothesis were correct, enzyme-treated cells cultured with 3-aminobenzamide to inhibit synthesis of poly(ADP-ribose) polymers would result in a significant increase in mutations outside the restriction enzyme sites. The spectrum of mutations observed after electroporation of PvuII (which produces blunt-end double-strand breaks) or PvuI (which produces cohesive-end double-strand breaks) was similar in untreated and 3-aminobenzamide-treated cells. Thus, our results do not support the hypothesis that the increase in damage observed when poly(ADP-ribosyl)ation is inhibited is due to a chaotic, nonspecific attack on DNA by endogenous cellular nucleases.     

Physical and functional interaction between DNA ligase IIIalpha and poly(ADP-Ribose) polymerase 1 in DNA single-strand break repair

The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase IIIalpha, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase IIIalpha but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase IIIalpha-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADP-ribose) polymer.     

ATP for the DNA ligation step in base excision repair is generated from poly(ADP-ribose)

In mammalian cells, the base excision repair (BER) pathway is the main route to counteract the mutagenic effects of DNA lesions. DNA nicks induce, among others, DNA polymerase activities and the synthesis of poly(ADP-ribose). It is shown here that poly(ADP-ribose) serves as an energy source for the final and rate-limiting step of BER, DNA ligation. This conclusion was drawn from experiments in which the fate of [(32)P]poly(ADP-ribose) or [(32)P]NAD added to HeLa nuclear extracts was systematically followed. ATP was synthesized from poly(ADP-ribose) in a pathway that strictly depended on nick-induced DNA synthesis. NAD was used for the synthesis of poly(ADP-ribose), which, in turn, was converted to ATP by pyrophosphorylytic cleavage utilizing the pyrophosphate generated from dNTPs during DNA synthesis. The adenylyl moiety was then preferentially used to adenylate DNA ligase III, from which it was transferred to the 5'-phosphoryl end of the nicked DNA. Finally, ligation to the 3'-OH end resulted in the release of AMP. When using NAD, but not poly(ADP-ribose), in the presence of 3-aminobenzamide, the entire process was blocked, confirming poly(ADP-ribosyl)ation to be the essential initial step. Thus, poly(ADP-ribose) polymerase-1, DNA polymerase beta, and ligase III interact with x-ray repair cross-complementing protein-1 within the BER complex, which ensures that ATP is generated and specifically used for DNA ligation.     

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.