PARP-1 and its associated nucleases in DNA damage response

Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) acts as a DNA damage sensor. It recognizes DNA damage and facilitates DNA repair by recruiting DNA repair machinery to damage sites. Recent studies reported that PARP-1 also plays an important role in DNA replication by recognizing the unligated Okazaki fragments and controlling the speed of fork elongation. On the other hand, emerging evidence reveals that excessive activation of PARP-1 causes chromatin DNA fragmentation and triggers an intrinsic PARP-1-dependent cell death program designated parthanatos, which can be blocked by genetic deletion or pharmacological inhibition of PARP-1. Therefore, PARP-1 plays an essential role in maintaining genomic stability by either facilitating DNA repair/replication or triggering DNA fragmentation to kill cells. A group of structure-specific nucleases is crucial for executing DNA incision and fragmentation following PARP-1 activation. In this review, we will discuss how PARP-1 coordinates with its associated nucleases to maintain genomic integrity and control the decision of cell life and death.     

Poly(ADP-ribose) polymerase: a guardian angel protecting the genome and suppressing tumorigenesis

Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage generated either exogenously or endogenously. This post-translational modification is catalyzed by poly(ADP-ribose) polymerase (PARP, PARP-1, EC 2.4.2.30). It is proposed that this protein plays a multifunctional role in many cellular processes, including DNA repair, recombination, cell proliferation and death, as well as genomic stability. Chemical inhibitors of the enzyme, dominant negative or null mutations of PARP-1 cause a high degree of genomic instability in cells. Inhibition of PARP activity by chemical inhibitors renders mice or rats susceptible to carcinogenic agents in various tumor models, indicating a role for PARP-1 in suppressing tumorigenesis. Despite the above observations, PARP-1 knockout mice are generally not prone to the development of tumors. An enhanced tumor development was observed, however, when the PARP-1 null mutation was introduced into severely compromised immune-deficient mice (a mutation in DNA-dependent protein kinase) or mice lacking other DNA repair or chromosomal guardian molecules, such as p53 or Ku80. These studies indicate that PARP-1 functions as a cofactor to suppress tumorigenesis via its role in stabilization of the genome, and/or by interacting with other DNA strand break-sensing molecules. Studies using PARP-1 mutants and chemical inhibitors have started to shed light on the role of this protein and of the specific protein post-translational modification in the control of genomic stability and hence its involvement in cancer.     

Poly(ADP-ribose) polymerase 1 (PARP-1) binds to 8-oxoguanine-DNA glycosylase (OGG1)

Human 8-oxoguanine-DNA glycosylase (OGG1) plays a major role in the base excision repair pathway by removing 8-oxoguanine base lesions generated by reactive oxygen species. Here we report a novel interaction between OGG1 and Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA-damage sensor protein involved in DNA repair and many other cellular processes. We found that OGG1 binds directly to PARP-1 through the N-terminal region of OGG1, and this interaction is enhanced by oxidative stress. Furthermore, OGG1 binds to PARP-1 through its BRCA1 C-terminal (BRCT) domain. OGG1 stimulated the poly(ADP-ribosyl)ation activity of PARP-1, whereas decreased poly(ADP-ribose) levels were observed in OGG1(-/-) cells compared with wild-type cells in response to DNA damage. Importantly, activated PARP-1 inhibits OGG1. Although the OGG1 polymorphic variant proteins R229Q and S326C bind to PARP-1, these proteins were defective in activating PARP-1. Furthermore, OGG1(-/-) cells were more sensitive to PARP inhibitors alone or in combination with a DNA-damaging agent. These findings indicate that OGG1 binding to PARP-1 plays a functional role in the repair of oxidative DNA damage.     

Human proliferating cell nuclear antigen,poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners

We addressed the analysis of the physical and functional association of proliferating cell nuclear antigen (PCNA), a protein involved in many DNA transactions, with poly(ADP-ribose) polymerase (PARP-1), an enzyme that plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. We demonstrated that PARP-1 and PCNA co-immunoprecipitated both from the soluble and the DNA-bound fraction isolated from S-phase-synchronized HeLa cells. Immunoprecipitation experiments with purified proteins further confirmed a physical association between PARP-1 and PCNA. To investigate the effect of this association on PARP-1 activity, an assay based on the incorporation of radioactive NAD was performed. Conversely, the effect of PARP-1 on PCNA-dependent DNA synthesis was assessed by a DNA polymerase delta assay. A marked inhibition of both reactions was found. Unexpectedly, PARP-1 activity also decreased in the presence of p21waf1/cip1. By pull-down experiments, we provided the first evidence for an association between PARP-1 and p21, which involves the C-terminal part of p21 protein. This association was further demonstrated to occur also in vivo in MNNG (N-methyl-N'-nitro-N-nitrosoguanidine)-treated human fibroblasts. These observations suggest that PARP-1 and p21 could cooperate in regulating the functions of PCNA during DNA replication/repair.     

Poly(ADP-ribose) Polymerase (PARP-1) in Homologous Recombination and as a Target for Cancer Therapy

Poly(ADP-ribose) polymerase (PARP-1) binds to DNA breaks to facilitate DNA repair. However, the role of PARP-1 in DNA repair appears to not be critical since PARP-1 knockout mice are viable, fertile and do not develop early onset tumours. Cells isolated from these mice show an increased level of homologous recombination. There is an intricate link between homologous recombination and PARP-1 and a possible role for PARP-1 in DNA double-strand break repair. Although PARP-1 appears not to be required for homologous recombination itself, it regulates the process through its involvement in the repair of DNA single-strand breaks (SSBs). SSBs persisting into the S phase of the cell cycle collapse replication forks, triggering homologous recombination for replication restart. We discuss the recent discoveries on the use of PARP-1 inhibitors as a targeted cancer therapy for recombination deficient cancers, such as BRCA2 tumours.     

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.     

The DNA topoisomerase IIbeta binding protein 1 (TopBP1) interacts with poly (ADP-ribose) polymerase (PARP-1)

We investigated the physical association of the DNA topoisomerase IIbeta binding protein 1 (TopBP1), involved in DNA replication and repair but also in regulation of apoptosis, with poly(ADP-ribose) polymerase-1 (PARP-1). This enzyme plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. It was shown that the sixth BRCA1 C-terminal (BRCT) domain of TopBP1 interacts with a protein fragment of PARP-1 in vitro containing the DNA-binding and the automodification domains. More significantly, the in vivo interaction of endogenous TopBP1 and PARP-1 proteins could be shown in HeLa-S3 cells by co-immunoprecipitation. TopBP1 and PARP-1 are localized within overlapping regions in the nucleus of HeLa-S3 cells as shown by immunofluorescence. Exposure to UVB light slightly enhanced the interaction between both proteins. Furthermore, TopBP1 was detected in nuclear regions where poly(ADP-ribose) (PAR) synthesis takes place and is ADP-ribosylated by PARP-1. Finally, cellular (ADP-ribosyl)ating activity impairs binding of TopBP1 to Myc-interacting zinc finger protein-1 (Miz-1). The results indicate an influence of post-translational modifications of TopBP1 on its function during DNA repair.     

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.     

The BRCT Domain of PARP-1 Is Required for Immunoglobulin Gene Conversion

Genetic variation at immunoglobulin (Ig) gene variable regions in B-cells is created through a multi-step process involving deamination of cytosine bases by activation-induced cytidine deaminase (AID) and their subsequent mutagenic repair. To protect the genome from dangerous, potentially oncogenic effects of off-target mutations, both AID activity and mutagenic repair are targeted specifically to the Ig genes. However, the mechanisms of targeting are unknown and recent data have highlighted the role of regulating mutagenic repair to limit the accumulation of somatic mutations resulting from the more widely distributed AID-induced lesions to the Ig genes. Here we investigated the role of the DNA damage sensor poly-(ADPribose)-polymerase-1 (PARP-1) in the repair of AID-induced DNA lesions. We show through sequencing of the diversifying Ig genes in PARP-1^−/− DT40 B-cells that PARP-1 deficiency results in a marked reduction in gene conversion events and enhanced high-fidelity repair of AID-induced lesions at both Ig heavy and light chains. To further characterize the role of PARP-1 in the mutagenic repair of AID-induced lesions, we performed functional analyses comparing the role of engineered PARP-1 variants in high-fidelity repair of DNA damage induced by methyl methane sulfonate (MMS) and the mutagenic repair of lesions at the Ig genes induced by AID. This revealed a requirement for the previously uncharacterized BRCT domain of PARP-1 to reconstitute both gene conversion and a normal rate of somatic mutation at Ig genes, while being dispensable for the high-fidelity base excision repair. From these data we conclude that the BRCT domain of PARP-1 is required to initiate a significant proportion of the mutagenic repair specific to diversifying antibody genes. This role is distinct from the known roles of PARP-1 in high-fidelity DNA repair, suggesting that the PARP-1 BRCT domain has a specialized role in assembling mutagenic DNA repair complexes involved in antibody diversification.     

Repair pathway for PARP-1 DNA-protein crosslinks

Poly(ADP-ribose) polymerase-1 (PARP-1) is a regulatory enzyme involved in many different processes of DNA and RNA metabolism, including DNA repair. Previously, PARP-1 was found capable of forming a covalent DNA-protein crosslink (DPC) at the apurinic/apyrimidinic (AP) site in double-stranded DNA. The C1´ atom of the AP site participates in Schiff base formation with a lysine side chain in PARP-1, and a covalent bond is formed upon reduction of the Schiff base. The PARP-1 DPC is formed in vivo where DPC formation correlates with AP site induction by a monofunctional alkylating agent. Here, we examined repair of PARP-1 DPCs in mouse fibroblasts and found that a proteasome inhibitor, MG-132, reduces repair resulting in accumulation of PARP-1 DPCs and increased alkylating agent cytotoxicity. Using a model DNA substrate mimicking the PARP-1 DPC after proteasomal degradation, we found that repair is completed by a sub-pathway of base excision repair (BER). Tyrosyl-DNA phosphodiesterase 1 was proficient in removing the ring-open AP site sugar at the phosphodiester linkage, leaving an intermediate for processing by other BER enzymes. The results reveal proteasomal degradation of the PARP-1 DPC is active in mouse fibroblasts and that a model repair intermediate is processed by the BER machinery.     

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.     

PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer.     

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.     

Increased PARP-1 association with DNA in alkylation damaged, PARP-inhibited mouse fibroblasts

Treatment of base excision repair-proficient mouse fibroblasts with the DNA alkylating agent methyl methanesulfonate (MMS) and a small molecule inhibitor of PARP-1 results in a striking cell killing phenotype, as previously reported. Earlier studies showed that the mechanism of cell death is apoptosis and requires DNA replication, expression of PARP-1, and an intact S-phase checkpoint cell signaling system. It is proposed that activity-inhibited PARP-1 becomes immobilized at DNA repair intermediates, and that this blocks DNA repair and interferes with DNA replication, eventually promoting an S-phase checkpoint and G(2)-M block. Here we report studies designed to evaluate the prediction that inhibited PARP-1 remains DNA associated in cells undergoing repair of alkylation-induced damage. Using chromatin immunoprecipitation with anti-PARP-1 antibody and qPCR for DNA quantification, a higher level of DNA was found associated with PARP-1 in cells treated with MMS plus PARP inhibitor than in cells without inhibitor treatment. These results have implications for explaining the extreme hypersensitivity phenotype after combination treatment with MMS and a PARP inhibitor.     

Poly(ADP-ribose) Contributes to an Association between Poly(ADP-ribose) Polymerase-1 and Xeroderma Pigmentosum Complementation Group A in Nucleotide Excision Repair

Exposure to ultraviolet radiation (UVR) promotes the formation of UVR-induced, DNA helix distorting photolesions such as (6-4) pyrimidine-pyrimidone photoproducts and cyclobutane pyrimidine dimers. Effective repair of such lesions by the nucleotide excision repair (NER) pathway is required to prevent DNA mutations and chromosome aberrations. Poly(ADP-ribose) polymerase-1 (PARP-1) is a zinc finger protein with well documented involvement in base excision repair. PARP-1 is activated in response to DNA damage and catalyzes the formation of poly(ADP-ribose) subunits that assist in the assembly of DNA repair proteins at sites of damage. In this study, we present evidence for PARP-1 contributions to NER, extending the knowledge of PARP-1 function in DNA repair beyond the established role in base excision repair. Silencing the PARP-1 protein or inhibiting PARP activity leads to retention of UVR-induced photolesions. PARP activation following UVR exposure promotes association between PARP-1 and XPA, a central protein in NER. Administration of PARP inhibitors confirms that poly(ADP-ribose) facilitates PARP-1 association with XPA in whole cell extracts, in isolated chromatin complexes, and in vitro. Furthermore, inhibition of PARP activity decreases UVR-stimulated XPA chromatin association, illustrating that these relationships occur in a meaningful context for NER. These results provide a mechanistic link for PARP activity in the repair of UVR-induced photoproducts.     

PARP-3 Is a Mono-ADP-ribosylase That Activates PARP-1 in the Absence of DNA

The PARP-3 protein is closely related to the PARP-1 and PARP-2 proteins, which are involved in DNA repair and genome maintenance. Here, we characterized the biochemical properties of human PARP-3. PARP-3 is able to ADP-ribosylate itself as well as histone H1, a previously unknown substrate for PARP-3. PARP-3 is not activated upon binding to DNA and is a mono-ADP-ribosylase, in contrast to PARP-1 and PARP-2. PARP-3 interacts with PARP-1 and activates PARP-1 in the absence of DNA, resulting in synthesis of polymers of ADP-ribose. The N-terminal WGR domain of PARP-3 is involved in this activation. The functional interaction between PARP-3 and PARP-1 suggests that it may have a role in DNA repair. However, here we report that PARP-3 small interfering RNA-depleted cells are not sensitive to the topoisomerase I poison camptothecin, inducing DNA single-strand breaks, and repair these lesions as efficiently as wild-type cells. Altogether, these results suggest that the interaction between PARP-1 and PARP-3 is unrelated to DNA single-strand break repair.     

Multiple roles for poly(ADP-ribose)polymerase-1 in neurological disease

Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear protein activated by DNA damage. PARP-1 activation is associated in DNA repair, cell death and inflammation. Since oxidative stress induced robust DNA damage and wide spread inflammatory responses are common pathologies of various CNS diseases, the interest toward PARP-1 as a therapeutic target has peaked. This review introduces mechanism of PARP-1 activation, the role of PARP-1 in cell physiology and pathology, and discusses the potential of PARP-1 inhibition as a therapy in acute and chronic CNS diseases.