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Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

ABSTRACT: Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

SUBMITTER: Robu M

PROVIDER: S-EPMC5565455 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Role of poly(ADP-ribose) polymerase-1 in the removal of UV-induced DNA lesions by nucleotide excision repair.

Project description:Among the earliest responses of mammalian cells to DNA damage is catalytic activation of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Activated PARP-1 forms the polymers of ADP-ribose (pADPr or PAR) that posttranslationally modify its target proteins, such as PARP-1 and DNA repair-related proteins. Although this metabolism is known to be implicated in other repair pathways, here we show its role in the versatile nucleotide excision repair pathway (NER) that removes a variety of DNA damages including those induced by UV. We show that PARP inhibition or specific depletion of PARP-1 decreases the efficiency of removal of UV-induced DNA damage from human skin fibroblasts or mouse epidermis. Using NER-proficient and -deficient cells and in vitro PARP-1 assays, we show that damaged DNA-binding protein 2 (DDB2), a key lesion recognition protein of the global genomic subpathway of NER (GG-NER), associates with PARP-1 in the vicinity of UV-damaged chromatin, stimulates its catalytic activity, and is modified by pADPr. PARP inhibition abolishes UV-induced interaction of DDB2 with PARP-1 or xeroderma pigmentosum group C (XPC) and also decreases localization of XPC to UV-damaged DNA, which is a key step that leads to downstream events in GG-NER. Thus, PARP-1 collaborates with DDB2 to increase the efficiency of the lesion recognition step of GG-NER.

| S-EPMC3562836 | biostudies-literature

Poly(ADP-ribose) contributes to an association between poly(ADP-ribose) polymerase-1 and xeroderma pigmentosum complementation group A in nucleotide excision repair.

Project description: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.

| S-EPMC3501068 | biostudies-literature

Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium.

Project description:Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; <10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations.

| S-EPMC4718819 | biostudies-literature

Impact of poly(ADP-Ribose) polymerase 1 on mitochondrial DNA repair

Project description:Formation of a repair enzyme complex is beneficial to DNA repair. Despite cellular studies showed that mitochondrial DNA polymerase (Pol ) and poly(ADP-ribose) polymerase 1 (PARP1) were found in the same complex along with other mitochondrial DNA repair enzymes and mitochondrial PARP1 level is correlated with mtDNA integrity, the molecular basis for the functional connection between Pol and PARP1 has not been illustrated, because cellular functions of PARP1 in DNA repair are intertwined with metabolism via NAD+ (nicotinamide adenosine dinucleotide), the substrate of PARP1 and a metabolic cofactor. To dissect the direct effect of PARP1 on mtDNA from the secondary perturbation metabolism, we report here biochemical studies that recapitulated Pol PARylation observed in cells, showed that PARP1 regulates Pol activity during DNA repair in a metabolic cofactor NAD+ (nicotinamide adenosine dinucleotide)-dependent manner. In the absence of NAD+, PARP1 completely inhibits Pol, while increasing NAD+ level to physiological concentration enables Pol to resume maximum repair activity. Because cellular NAD+ levels are linked to metabolism and to ATP production via oxidative phosphorylation, our results suggest that mtDNA damage repair is correlated with cellular metabolic state and integrity of the respiratory chain.

2020-12-16 | MSV000086606 | MassIVE

p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates.

Project description:The cell cycle inhibitor p21CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates containing single-strand breaks, we found that full-length recombinant GST-tagged p21 but not a C-terminal domain truncated form of p21 was able to stimulate the PARP-1 binding to BER intermediates with no significant influence on the catalytic activity of PARP-1. In addition, we investigate whether the activation of PARP-1 through poly(ADP-ribose) (PAR) synthesis, is required for its interaction with p21. We have found that in human fibroblasts and in HeLa cells treated with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the interaction of p21 with PARP-1 was greatly dependent on PAR synthesis. In fact, an anti-PAR antibody was able to co-immunoprecipitate p21 and PARP-1 from extracts of MNNG-treated cells, while blocking PAR synthesis with the PARP-1 inhibitor Olaparib, drastically reduced the amount of p21 co-immunoprecipitated by a PARP-1 antibody. Our results provide the first evidence that p21 can stimulate the binding of PARP-1 to DNA repair intermediates, and that this cooperation requires PAR synthesis.

| S-EPMC4701469 | biostudies-literature

Poly(ADP-ribose) polymerase 1 regulates mitochondrial DNA repair in an NAD-dependent manner.

Project description:Mitochondrial DNA is located in organelle that house essential metabolic reactions and contains high reactive oxygen species. Therefore, mitochondrial DNA suffers more oxidative damage than its nuclear counterpart. Formation of a repair enzyme complex is beneficial to DNA repair. Recent studies have shown that mitochondrial DNA polymerase (Pol γ) and poly(ADP-ribose) polymerase 1 (PARP1) were found in the same complex along with other mitochondrial DNA repair enzymes, and mitochondrial PARP1 level is correlated with mtDNA integrity. However, the molecular basis for the functional connection between Pol γ and PARP1 has not yet been elucidated because cellular functions of PARP1 in DNA repair are intertwined with metabolism via NAD+ (nicotinamide adenosine dinucleotide), the substrate of PARP1, and a metabolic cofactor. To dissect the direct effect of PARP1 on mtDNA from the secondary perturbation of metabolism, we report here biochemical studies that recapitulated Pol γ PARylation observed in cells and showed that PARP1 regulates Pol γ activity during DNA repair in a metabolic cofactor NAD+ (nicotinamide adenosine dinucleotide)-dependent manner. In the absence of NAD+, PARP1 completely inhibits Pol γ, while increasing NAD+ levels to a physiological concentration that enables Pol γ to resume maximum repair activity. Because cellular NAD+ levels are linked to metabolism and to ATP production via oxidative phosphorylation, our results suggest that mtDNA damage repair is coupled to cellular metabolic state and the integrity of the respiratory chain.

| S-EPMC7949115 | biostudies-literature

Nucleotide excision repair of abasic DNA lesions.

Project description:Apurinic/apyrimidinic (AP) sites are a class of highly mutagenic and toxic DNA lesions arising in the genome from a number of exogenous and endogenous sources. Repair of AP lesions takes place predominantly by the base excision pathway (BER). However, among chemically heterogeneous AP lesions formed in DNA, some are resistant to the endonuclease APE1 and thus refractory to BER. Here, we employed two types of reporter constructs accommodating synthetic APE1-resistant AP lesions to investigate the auxiliary repair mechanisms in human cells. By combined analyses of recovery of the transcription rate and suppression of transcriptional mutagenesis at specifically positioned AP lesions, we demonstrate that nucleotide excision repair pathway (NER) efficiently removes BER-resistant AP lesions and significantly enhances the repair of APE1-sensitive ones. Our results further indicate that core NER components XPA and XPF are equally required and that both global genome (GG-NER) and transcription coupled (TC-NER) subpathways contribute to the repair.

| S-EPMC6895268 | biostudies-literature

DNA Repair Enzyme Poly(ADP-Ribose) Polymerase 1/2 (PARP1/2)-Targeted Nuclear Imaging and Radiotherapy.

Project description:Since it was discovered that many tumor types are vulnerable to inhibition of the DNA repair machinery, research towards efficient and selective inhibitors has accelerated. Amongst other enzymes, poly(ADP-ribose)-polymerase 1 (PARP1) was identified as a key player in this process, which resulted in the development of selective PARP inhibitors (PARPi) as anti-cancer drugs. Most small molecule PARPi's exhibit high affinity for both PARP1 and PARP2. PARPi are under clinical investigation for mono- and combination therapy in several cancer types and five PARPi are now clinically approved. In parallel, radiolabeled PARPi have emerged for non-invasive imaging of PARP1 expression. PARP imaging agents have been suggested as companion diagnostics, patient selection, and treatment monitoring tools to improve the outcome of PARPi therapy, but also as stand-alone diagnostics. We give a comprehensive overview over the preclinical development of PARP imaging agents, which are mostly based on the PARPi olaparib, rucaparib, and recently also talazoparib. We also report on the current status of clinical translation, which involves a growing number of early phase trials. Additionally, this work provides an insight into promising approaches of PARP-targeted radiotherapy based on Auger and α-emitting isotopes. Furthermore, the review covers synthetic strategies for PARP-targeted imaging and therapy agents that are compatible with large scale production and clinical translation.

| S-EPMC8909184 | biostudies-literature

Inhibition of poly(ADP-ribose) polymerase-1 by arsenite interferes with repair of oxidative DNA damage.

Project description:Arsenic enhances skin tumor formation when combined with other carcinogens, including UV radiation (UVR). In this study we report that low micromolar concentrations of arsenite synergistically increases UVR-induced oxidative DNA damage in human keratinocytes as detected by 8-hydroxyl-2'-deoxyguanine (8-OHdG) formation. Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in base excision repair, a process that repairs 8-OHdG lesions. Arsenite suppresses UVR-induced PARP-1 activation in a concentration-dependent manner. Inhibition of PARP-1 activity by 3-aminobenzamide or small interfering RNA silencing of PARP-1 expression significantly increases UVR-induced 8-OHdG formation, suggesting that inhibition of PARP-1 activity by arsenite contributes to oxidative DNA damage. PARP-1 is a zinc finger protein, and mass spectrometry analysis reveals that arsenite can occupy a synthetic apopeptide representing the first zinc finger of PARP-1 (PARPzf). When the PARPzf peptide is preincubated with Zn(II) followed by incubation with increasing concentrations of arsenite, the ZnPARPzf signal is decreased while the AsPARPzf signal intensity is increased as a function of arsenite dose, suggesting a competition between zinc and arsenite for the same binding site. Addition of Zn(II) abolished arsenite enhancement of UVR-stimulated 8-OHdG generation and restored PARP-1 activity. Our findings demonstrate that arsenite inhibits oxidative DNA damage repair and suggest that interaction of arsenite with the PARP-1 zinc finger domain contributes to the inhibition of PARP-1 activity by arsenite. Arsenite inhibition of poly(ADP-ribosyl)ation is one likely mechanism for the reported co-carcinogenic activities of arsenic in UVR-induced skin carcinogenesis.

| S-EPMC2652344 | biostudies-literature

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

Project description: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.

| S-EPMC3082910 | biostudies-literature

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