RESUMEN
In this issue of Cell Metabolism, Li et al. report that the highly expressed aldehyde dehydrogenase 1 family member A3 interacts with pyruvate kinase M2 (PKM2) in glioblastoma cells. Consequently, PKM2 tetramerization and activation promote lactate production, leading to the lactylation and nuclear translocation of XRCC1 for DNA damage repair and therapeutic resistance.
Asunto(s)
Daño del ADN , Reparación del ADN , Humanos , Glioblastoma/metabolismo , Glioblastoma/patología , Glioblastoma/genética , Efecto Warburg en Oncología , Proteínas de Unión al ADN/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Proteínas de Unión a Hormona Tiroide , Hormonas Tiroideas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Portadoras/metabolismo , Aldehído Deshidrogenasa/metabolismo , Aldehído Deshidrogenasa/genéticaRESUMEN
Patients with high ALDH1A3-expressing glioblastoma (ALDH1A3hi GBM) show limited benefit from postoperative chemoradiotherapy. Understanding the mechanisms underlying such resistance in these patients is crucial for the development of new treatments. Here, we show that the interaction between ALDH1A3 and PKM2 enhances the latter's tetramerization and promotes lactate accumulation in glioblastoma stem cells (GSCs). By scanning the lactylated proteome in lactate-accumulating GSCs, we show that XRCC1 undergoes lactylation at lysine 247 (K247). Lactylated XRCC1 shows a stronger affinity for importin α, allowing for greater nuclear transposition of XRCC1 and enhanced DNA repair. Through high-throughput screening of a small-molecule library, we show that D34-919 potently disrupts the ALDH1A3-PKM2 interaction, preventing the ALDH1A3-mediated enhancement of PKM2 tetramerization. In vitro and in vivo treatment with D34-919 enhanced chemoradiotherapy-induced apoptosis of GBM cells. Together, our findings show that ALDH1A3-mediated PKM2 tetramerization is a potential therapeutic target to improve the response to chemoradiotherapy in ALDH1A3hi GBM.
Asunto(s)
Glioblastoma , Proteínas de Unión a Hormona Tiroide , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X , Glioblastoma/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Animales , Línea Celular Tumoral , Ratones , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Proteínas de la Membrana/metabolismo , Proteínas Portadoras/metabolismo , Hormonas Tiroideas/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Aldehído Oxidorreductasas , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NHRESUMEN
Non-membrane compartments or biomolecular condensates play an important role in the regulation of cellular processes including DNA repair. Here, an ability of XRCC1, a scaffold protein involved in DNA base excision repair (BER) and single-strand break repair, to form protein-rich microphases in the presence of DNA duplexes was discovered. We also showed that the gap-filling activity of BER-related DNA polymerase λ (Pol λ) is significantly increased by the presence of XRCC1. The stimulation of the Pol λ activity was observed only at micromolar XRCC1 concentrations, which were well above the nanomolar dissociation constant determined for the XRCC1-Pol λ complex and pointed to the presence of an auxiliary stimulatory factor in addition to protein-protein interactions. Indeed, according to dynamic light scattering measurements, the stimulation of the Pol λ activity by XRCC1 was coupled with microphase separation in a protein-DNA mixture. Fluorescence microscopy revealed colocalization of Pol λ, XRCC1, and gapped DNA within the microphases. Thus, stimulation of Pol λ activity is caused both by its interaction with XRCC1 and by specific conditions of microphase separation; this phenomenon is shown for the first time.
Asunto(s)
ADN Polimerasa beta , Reparación del ADN , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , ADN Polimerasa beta/metabolismo , Humanos , ADN/metabolismo , Unión ProteicaRESUMEN
TIMELESS (TIM) in the fork protection complex acts as a scaffold of the replisome to prevent its uncoupling and ensure efficient DNA replication fork progression. Nevertheless, its underlying basis for coordinating leading and lagging strand synthesis to limit single-stranded DNA (ssDNA) exposure remains elusive. Here, we demonstrate that acute degradation of TIM at ongoing DNA replication forks induces the accumulation of ssDNA gaps stemming from defective Okazaki fragment (OF) processing. Cells devoid of TIM fail to support the poly(ADP-ribosyl)ation necessary for backing up the canonical OF processing mechanism mediated by LIG1 and FEN1. Consequently, recruitment of XRCC1, a known effector of PARP1-dependent single-strand break repair, to post-replicative ssDNA gaps behind replication forks is impaired. Physical disruption of the TIM-PARP1 complex phenocopies the rapid loss of TIM, indicating that the TIM-PARP1 interaction is critical for the activation of this compensatory pathway. Accordingly, combined deficiency of FEN1 and the TIM-PARP1 interaction leads to synergistic DNA damage and cytotoxicity. We propose that TIM is essential for the engagement of PARP1 to the replisome to coordinate lagging strand synthesis with replication fork progression. Our study identifies TIM as a synthetic lethal target of OF processing enzymes that can be exploited for cancer therapy.
Asunto(s)
Proteínas de Ciclo Celular , Replicación del ADN , ADN de Cadena Simple , Péptidos y Proteínas de Señalización Intracelular , Poli(ADP-Ribosa) Polimerasa-1 , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , ADN/metabolismo , ADN/genética , ADN Ligasa (ATP)/metabolismo , ADN Ligasa (ATP)/genética , Reparación del ADN , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Endonucleasas de ADN Solapado/metabolismo , Endonucleasas de ADN Solapado/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genéticaRESUMEN
OBJECTIVES: Reportedly, ganoderic acid A (GA-A) increases the sensitivity of hepatocellular carcinoma cells to cisplatin (DDP) chemotherapy. Therefore, this study aims to fathom the influence of GA-A on lung cancer cells. METHODS: After the construction of A549/DDP cells through exposure to DDP, the effects of GA-A on A549 and A549/DDP cells were revealed by cellular functional assays, western blot and quantitative reverse transcription PCR (qRT-PCR). The DDP-resistant lung cancer tumor was established in vivo, followed by further validation of the mechanism of GA-A. RESULTS: GA-A suppressed the viability, migration, and invasion while downregulating Beclin and autophagy marker LC3II/LC3I levels and upregulating P62 levels in A549 and A549/DDP cells. These effects were reversed by circFLNA overexpression. Also, GA-A reinforced the sensitivity of A549/DDP cells to DDP, elevated the apoptosis and regulated the circFLNA/miR-486-3p/cytochrome P450 family 1 subfamily A member 1 (CYP1A1)/X-ray repair cross-complementing 1 (XRCC1) axis. The reversal effects of circFLNA overexpression on GA-A-induced viability and apoptosis of A549/DDP cells could all be counteracted in the presence of 3MA. GA-A inhibited lung cancer tumor growth and blocked autophagy. CONCLUSION: GA-A suppresses autophagy by regulating the circFLNA/miR-486-3p/CYP1A1/XRCC1 axis to strengthen the sensitivity of lung cancer cells to DDP.
Asunto(s)
Antineoplásicos , Autofagia , Carcinoma de Pulmón de Células no Pequeñas , Ácidos Heptanoicos , Lanosterol , Neoplasias Pulmonares , MicroARNs , Humanos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Autofagia/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Cisplatino/farmacología , Citocromo P-450 CYP1A1/efectos de los fármacos , Citocromo P-450 CYP1A1/metabolismo , Resistencia a Antineoplásicos , Ácidos Heptanoicos/farmacología , Ácidos Heptanoicos/uso terapéutico , Lanosterol/análogos & derivados , Lanosterol/farmacología , Lanosterol/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , MicroARNs/efectos de los fármacos , MicroARNs/metabolismo , ARN Circular/efectos de los fármacos , ARN Circular/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/efectos de los fármacos , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
The joining of breaks in the DNA phosphodiester backbone is essential for genome integrity. Breaks are generated during normal processes such as DNA replication, cytosine demethylation during differentiation, gene rearrangement in the immune system and germ cell development. In addition, they are generated either directly by a DNA damaging agent or indirectly due to damage excision during repair. Breaks are joined by a DNA ligase that catalyzes phosphodiester bond formation at DNA nicks with 3' hydroxyl and 5' phosphate termini. Three human genes encode ATP-dependent DNA ligases. These enzymes have a conserved catalytic core consisting of three subdomains that encircle nicked duplex DNA during ligation. The DNA ligases are targeted to different nuclear DNA transactions by specific protein-protein interactions. Both DNA ligase IIIα and DNA ligase IV form stable complexes with DNA repair proteins, XRCC1 and XRCC4, respectively. There is functional redundancy between DNA ligase I and DNA ligase IIIα in DNA replication, excision repair and single-strand break repair. Although DNA ligase IV is a core component of the major double-strand break repair pathway, non-homologous end joining, the other enzymes participate in minor, alternative double-strand break repair pathways. In contrast to the nucleus, only DNA ligase IIIα is present in mitochondria and is essential for maintaining the mitochondrial genome. Human immunodeficiency syndromes caused by mutations in either LIG1 or LIG4 have been described. Preclinical studies with DNA ligase inhibitors have identified potentially targetable abnormalities in cancer cells and evidence that DNA ligases are potential targets for cancer therapy.
Asunto(s)
ADN Ligasas , Reparación del ADN , ADN , Animales , Humanos , ADN/genética , ADN/metabolismo , Daño del ADN , ADN Ligasa (ATP)/genética , ADN Ligasas/genética , ADN Ligasas/metabolismo , Replicación del ADN , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
Mammalian Base Excision Repair (BER) DNA ligases I and IIIα (LigI, LigIIIα) are major determinants of DNA repair fidelity, alongside with DNA polymerases. Here we compared activities of human LigI and LigIIIα on specific and nonspecific substrates representing intermediates of distinct BER sub-pathways. The enzymes differently discriminate mismatches in the nicked DNA, depending on their identity and position, but are both more selective against the 3'-end non-complementarity. LigIIIα is less active than LigI in premature ligation of one-nucleotide gapped DNA and more efficiently discriminates misinsertion products of DNA polymerase ß-catalyzed gap filling, that reinforces a leading role of LigIIIα in the accuracy of short-patch BER. LigI and LigIIIα reseal the intermediate of long-patch BER containing an incised synthetic AP site (F) with different efficiencies, depending on the DNA sequence context, 3'-end mismatch presence and coupling of the ligation reaction with DNA repair synthesis. Processing of this intermediate in the absence of flap endonuclease 1 generates non-canonical DNAs with bulged F site, which are very inefficiently repaired by AP endonuclease 1 and represent potential mutagenic repair products. The extent of conversion of the 5'-adenylated intermediates of specific and nonspecific substrates is revealed to depend on the DNA sequence context; a higher sensitivity of LigI to the sequence is in line with the enzyme structural feature of DNA binding. LigIIIα exceeds LigI in generation of potential abortive ligation products, justifying importance of XRCC1-mediated coordination of LigIIIα and aprataxin activities for the efficient DNA repair.
Asunto(s)
ADN Polimerasa beta , Reparación del ADN , Animales , Humanos , ADN/genética , ADN/metabolismo , ADN Polimerasa beta/genética , ADN Polimerasa beta/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Ligasas/genética , ADN Ligasas/metabolismo , Reparación por Escisión , Mamíferos/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
UV irradiation induces "bulky" DNA photodimers such as (6-4)-photoproducts and cyclobutane pyrimidine dimers that are removed by nucleotide excision repair, a complex process defective in the sunlight-sensitive and cancer-prone disease xeroderma pigmentosum. Some bacteria and lower eukaryotes can also repair photodimers by enzymatically simpler mechanisms, but such pathways have not been reported in normal human cells. Here, we have identified such a mechanism. We show that normal human cells can employ a DNA base excision repair process involving NTH1, APE1, PARP1, XRCC1, and FEN1 to rapidly remove a subset of photodimers at early times following UVC irradiation. Loss of these proteins slows the early rate of repair of photodimers in normal cells, ablates their residual repair in xeroderma pigmentosum cells, and increases UVC sensitivity â¼2-fold. These data reveal that human cells can excise photodimers using a long-patch base excision repair process that functions additively but independently of nucleotide excision repair.
Asunto(s)
Xerodermia Pigmentosa , Humanos , Xerodermia Pigmentosa/genética , Reparación del ADN/genética , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , Daño del ADN/genética , ADN/genética , Rayos Ultravioleta , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
T-2 mycotoxin is the most potent representative of the trichothecene group A and is produced by various Fusarium species, including F. sporotrichioides, F. poae, and F. acuminatum. T-2 toxin has been reported to have toxic effects on various tissues and organs, and humans and animals alike suffer a variety of pathological conditions after consumption of mycotoxin-contaminated food. The T-2 toxin's unique feature is dermal toxicity, characterized by skin inflammation. In this in vitro study, we investigated the molecular mechanism of T-2 toxin-induced genotoxicity in the human skin fibroblast-Hs68 cell line. For the purpose of investigation, the cells were treated with T-2 toxin in 0.1, 1, and 10 µM concentrations and incubated for 24 h and 48 h. Nuclear DNA (nDNA) is found within the nucleus of eukaryotic cells and has a double-helix structure. nDNA encodes the primary structure of proteins, consisting of the basic amino acid sequence. The alkaline comet assay results showed that T-2 toxin induces DNA alkali-labile sites. The DNA strand breaks in cells, and the DNA damage level is correlated with the increasing concentration and time of exposure to T-2 toxin. The evaluation of nDNA damage revealed that exposure to toxin resulted in an increasing lesion frequency in Hs68 cells with HPRT1 and TP53 genes. Further analyses were focused on mRNA expression changes in two groups of genes involved in the inflammatory and repair processes. The level of mRNA increased for all examined inflammatory genes (TNF, INFG, IL1A, and IL1B). In the second group of genes related to the repair process, changes in expression induced by toxin in genes-LIG3 and APEX were observed. The level of mRNA for LIG3 decreased, while that for APEX increased. In the case of LIG1, FEN, and XRCC1, no changes in mRNA level between the control and T-2 toxin probes were observed. In conclusion, the results of this study indicate that T-2 toxin shows genotoxic effects on Hs68 cells, and the molecular mechanism of this toxic effect is related to nDNA damage.
Asunto(s)
Micotoxinas , Toxina T-2 , Animales , Humanos , Micotoxinas/toxicidad , Micotoxinas/metabolismo , Toxina T-2/toxicidad , Toxina T-2/metabolismo , Línea Celular , Daño del ADN , ADN/metabolismo , Fibroblastos/metabolismo , ARN Mensajero/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
Bone marrow-derived human mesenchymal stem cells (hMSCs) can differentiate into various lineages, such as chondrocytes, adipocytes, osteoblasts, and neuronal lineages. It has been shown that the high-efficiency DNA-repair capacity of hMSCs is decreased during their differentiation. However, the underlying its mechanism during adipogenesis and osteogenesis is unknown. Herein, we investigated how alkyl-damage repair is modulated during adipogenic and osteogenic differentiation, especially focusing on the base excision repair (BER) pathway. Response to an alkylation agent was assessed via quantification of the double-strand break (DSB) foci and activities of BER-related enzymes during differentiation in hMSCs. Adipocytes showed high resistance against methyl methanesulfonate (MMS)-induced alkyl damage, whereas osteoblasts were more sensitive than hMSCs. During the differentiation, activities, and protein levels of uracil-DNA glycosylase were found to be regulated. In addition, ligation-related proteins, such as X-ray repair cross-complementing protein 1 (XRCC1) and DNA polymerase ß, were upregulated in adipocytes, whereas their levels and recruitment declined during osteogenesis. These modulations of BER enzyme activity during differentiation influenced DNA repair efficiency and the accumulation of DSBs as repair intermediates in the nucleus. Taken together, we suggest that BER enzymatic activity is regulated in adipogenic and osteogenic differentiation and these alterations in the BER pathway led to different responses to alkyl damage from those in hMSCs.
Asunto(s)
Adipogénesis , Células Madre Mesenquimatosas , Humanos , Adipogénesis/genética , Osteogénesis/fisiología , Médula Ósea/metabolismo , Células Cultivadas , Diferenciación Celular/fisiología , Reparación del ADN , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
DNA single-strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair-defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5-chloro-2'-deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA-defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility.
Asunto(s)
Antineoplásicos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Rotura Cromosómica , Reparación del ADN , Replicación del ADN , ADN , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
Efficient repair of oxidized DNA is critical for genome-integrity maintenance. Cockayne syndrome protein B (CSB) is an ATP-dependent chromatin remodeler that collaborates with Poly(ADP-ribose) polymerase I (PARP1) in the repair of oxidative DNA lesions. How these proteins integrate during DNA repair remains largely unknown. Here, using chromatin co-fractionation studies, we demonstrate that PARP1 and PARP2 promote recruitment of CSB to oxidatively-damaged DNA. CSB, in turn, contributes to the recruitment of XRCC1, and histone PARylation factor 1 (HPF1), and promotes histone PARylation. Using alkaline comet assays to monitor DNA repair, we found that CSB regulates single-strand break repair (SSBR) mediated by PARP1 and PARP2. Strikingly, CSB's function in SSBR is largely bypassed when transcription is inhibited, suggesting CSB-mediated SSBR occurs primarily at actively transcribed DNA regions. While PARP1 repairs SSBs at sites regardless of the transcription status, we found that PARP2 predominantly functions in actively transcribed DNA regions. Therefore, our study raises the hypothesis that SSBR is executed by different mechanisms based on the transcription status.
Asunto(s)
Cromatina , Humanos , Proteínas Portadoras/genética , Cromatina/genética , ADN/genética , ADN/metabolismo , Reparación del ADN , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
Laser micro-irradiation across the nucleus rapidly generates localized chromatin-associated DNA lesions permitting analysis of repair protein recruitment in living cells. Recruitment of three fluorescently-tagged base excision repair factors [DNA polymerase ß (pol ß), XRCC1 and PARP1], known to interact with one another, was compared in gene-deleted mouse embryonic fibroblasts and in those expressing the endogenous factor. A low energy micro-irradiation (LEMI) forming direct single-strand breaks and a moderate energy (MEMI) protocol that additionally creates oxidized bases were compared. Quantitative characterization of repair factor recruitment and sensitivity to clinical PARP inhibitors (PARPi) was dependent on the micro-irradiation protocol. PARP1 recruitment was biphasic and generally occurred prior to pol ß and XRCC1. After LEMI, but not after MEMI, pol ß and XRCC1 recruitment was abolished by the PARPi veliparib. Consistent with this, pol ß and XRCC1 recruitment following LEMI was considerably slower in PARP1-deficient cells. Surprisingly, the recruitment half-times and amplitudes for pol ß were less affected by PARPi than were XRCC1 after MEMI suggesting there is a XRCC1-independent component for pol ß recruitment. After LEMI, but not MEMI, pol ß dissociation was more rapid than that of XRCC1. Unexpectedly, PARP1 dissociation was slowed in the absence of XRCC1 as well with a PARPi after LEMI but not MEMI, suggesting that XRCC1 facilitates PARP1 dissociation from specific DNA lesions. XRCC1-deficient cells showed pronounced hypersensitivity to the PARPi talazoparib correlating with its known cytotoxic PARP1 trapping activity. In contrast to DNA methylating agents, PARPi only minimally sensitized pol ß and XRCC1-deficient cells to oxidative DNA damage suggesting differential binding of PARP1 to alternate repair intermediates. In summary, pol ß, XRCC1, and PARP1 display recruitment kinetics that exhibit correlated and unique properties that depend on the DNA lesion and PARP activity revealing that there are multiple avenues utilized in the repair of chromatin-associated DNA.
Asunto(s)
Reparación del ADN , Fibroblastos , Animales , Ratones , Fibroblastos/metabolismo , Daño del ADN , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , ADN/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Cromatina , Inhibidores de Poli(ADP-Ribosa) PolimerasasRESUMEN
DNA ligase I (LigI), the predominant enzyme that joins Okazaki fragments, interacts with PCNA and Pol δ. LigI also interacts with UHRF1, linking Okazaki fragment joining with DNA maintenance methylation. Okazaki fragments can also be joined by a relatively poorly characterized DNA ligase IIIα (LigIIIα)-dependent backup pathway. Here we examined the effect of LigI-deficiency on proteins at the replication fork. Notably, LigI-deficiency did not alter the kinetics of association of the PCNA clamp, the leading strand polymerase Pol ε, DNA maintenance methylation proteins and core histones with newly synthesized DNA. While the absence of major changes in replication and methylation proteins is consistent with the similar proliferation rate and DNA methylation levels of the LIG1 null cells compared with the parental cells, the increased levels of LigIIIα/XRCC1 and Pol δ at the replication fork and in bulk chromatin indicate that there are subtle replication defects in the absence of LigI. Interestingly, the non-replicative histone H1 variant, H1.0, is enriched in the chromatin of LigI-deficient mouse CH12F3 and human 46BR.1G1 cells. This alteration was not corrected by expression of wild type LigI, suggesting that it is a relatively stable epigenetic change that may contribute to the immunodeficiencies linked with inherited LigI-deficiency syndrome.
Asunto(s)
ADN Ligasa (ATP) , Replicación del ADN , Histonas , Antígeno Nuclear de Célula en Proliferación , Animales , Humanos , Ratones , Cromatina/genética , ADN/metabolismo , ADN Ligasa (ATP)/genética , ADN Ligasa (ATP)/metabolismo , ADN Ligasas/genética , ADN Ligasas/metabolismo , ADN Polimerasa III/genética , Histonas/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismoRESUMEN
Aprataxin (APTX), the product of the causative gene for hereditary neurogenerative syndromes Ataxia-oculomotor apraxia 1 and early onset ataxia with oculomotor apraxia and hypoalbuminemia, has an enzymatic activity of removing adenosine monophosphate from DNA 5'-end, which arises from abortive ligation by DNA ligases. It is also reported that APTX physically binds to XRCC1 and XRCC4, suggesting its involvement in DNA single-strand break repair (SSBR) and DNA double-strand break repair (DSBR) via non-homologous end joining pathway. Although the involvement of APTX in SSBR in association with XRCC1 has been established, the significance of APTX in DSBR and its interaction with XRCC4 have remained unclear. Here, we generated APTX knock-out (APTX-/-) cell from human osteosarcoma U2OS through CRISPR/Cas9-mediated genome editing system. APTX-/- cells exhibited increased sensitivity toward ionizing radiation (IR) and Camptothecin in association with retarded DSBR, as shown by increased number of retained γH2AX foci. However, the number of retained 53BP1 foci in APTX-/- cell was not discernibly different from wild-type cells, in stark contrast to XRCC4-depleted cells. The recruitment of GFP-tagged APTX (GFP-APTX) to the DNA damage sites was examined by laser micro-irradiation and live-cell imaging analysis using confocal microscope. The accumulation of GFP-APTX on the laser track was attenuated by siRNA-mediated depletion of XRCC1, but not XRCC4. Moreover, the deprivation of APTX and XRCC4 displayed additive inhibitory effects on DSBR after IR exposure and end joining of GFP reporter. These findings collectively suggest that APTX acts in DSBR in a manner distinct from XRCC4.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN , Humanos , Ataxia Cerebelosa , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
Base excision repair (BER) is aimed at repair of damaged bases, which are the largest group of DNA lesions. The main steps of BER are recognition and removal of the aberrant base, cutting of the DNA sugar-phosphate backbone, gap processing (including dNMP insertion), and DNA ligation. The precise function of BER depends on the regulation of each step by regulatory/accessory proteins, the most important of which is poly(ADP-ribose) (PAR) polymerase 1 (PARP1). PARP1 plays an important role in DNA repair, maintenance of genome integrity, and regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. There is no systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells. Whole-cell extracts and RNA preparations obtained from the parental HEK293T cell line and its derivative HEK293T/P1-KD cell line with reduced PARP1 expression (shPARP1-expressing cells, a PARP1 knockdown) were used to assess the levels of mRNAs coding for BER proteins: PARP1, PARP2, uracil DNA glycosylase (UNG2), AP endonuclease 1 (APE1), DNA polymerase ß (POLß), DNA ligase III (LIG3), and XRCC1. Catalytic activities of the enzymes were evaluated in parallel. No significant effect of the PARP1 content was observed for the mRNA levels of UNG2, APE1, POLß, LIG3, and XRCC1. The amount of the PARP2 mRNA proved to be reduced two times in HEK293T/P1-KD cells. Activities of these enzymes in whole-cell extracts did not differ significantly between HEK293T and HEK293T/P1-KD cells. No significant change was observed in the efficiencies of the reactions catalyzed by UNG2, APE1, POLß, and LIG3 in conditions of PAR synthesis. A DNA PARylation pattern did not dramatically change in a HEK293T/P1-KD cell extract with a reduced PARP1 content as compared with an extract of the parental HEK293T cell line.
Asunto(s)
Reparación del ADN , Poli(ADP-Ribosa) Polimerasas , Humanos , Extractos Celulares , Células HEK293 , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Reparación del ADN/genética , ADN/genética , Daño del ADN , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
DNA Polymerase ß (Polß) performs two critical enzymatic steps during base excision repair (BER) - gap filling (nucleotidyl transferase activity) and gap tailoring (dRP lyase activity). X-ray repair cross complementing 1 (XRCC1) facilitates the recruitment of Polß to sites of DNA damage through an evolutionarily conserved Polß/XRCC1 interaction interface, the V303 loop. While previous work describes the importance of the Polß/XRCC1 interaction for human Polß protein stability and recruitment to sites of DNA damage, the impact of disrupting the Polß/XRCC1 interface on animal viability, physiology, and fertility is unknown. Here, we characterized the effect of disrupting Polß/XRCC1 heterodimerization in mice and mouse cells by complimentary approaches. First, we demonstrate, via laser micro-irradiation, that mouse Polß amino acid residues L301 and V303 are critical to facilitating Polß recruitment to sites of DNA damage. Next, we solved the crystal structures of mouse wild type Polß and a mutant protein harboring alterations in residues L301 and V303 (L301R/V303R). Our structural analyses suggest that Polß amino acid residue V303 plays a role in maintaining an interaction with the oxidized form of XRCC1. Finally, we created CRISPR/Cas9-modified Polb mice with homozygous L301R/V303R mutations (PolbL301R-V303R/L301R-V303R) that are fertile yet exhibit 15% reduced body weight at 17 weeks of age, as compared to heterozygous mice. Fibroblasts derived from PolbL301R-V303R/L301R-V303R mice demonstrate that mutation of mouse Polß's XRCC1 interaction domain leads to an â¼85% decrease in Polß protein levels. In all, these studies are consistent with a role for the oxidized form of XRCC1 in providing stability to the Polß protein through Polß/XRCC1 heterodimer formation.
Asunto(s)
ADN Polimerasa beta , Proteínas de Unión al ADN , Animales , Ratones , Aminoácidos/genética , Daño del ADN , ADN Polimerasa beta/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Fertilidad , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
PARP1 (aka ARTD1) acts as a prime sensor of cellular genotoxic stress response. PARP1 detects DNA strand breaks and subsequently catalyzes the formation of poly(ADP-ribose) (PAR), which leads to the recruitment of the scaffold protein XRCC1 during base excision and single strand break repair and the assembly of multi-protein complexes to promote DNA repair. Here, we reveal that the recruitment of either protein to sites of DNA damage is impeded in the absence of the other, indicating a strong reciprocal relationship between the two DNA repair factors during genotoxic stress response. We further analyzed several cellular and molecular endpoints in HeLa PARP1 KO, XRCC1 KO, and PARP1/XRCC1 double KO (DKO) cells after genotoxic treatments, i.e., PARylation response, NAD+ levels, clonogenic survival, cell cycle progression, cell death, and DNA repair. The analysis of NAD+ levels and cytotoxicity after treatment with the topoisomerase I inhibitor camptothecin revealed a hypersensitivity phenotype of XRCC1 KO cells compared to PARP1 KO cells-an effect that could be rescued by the additional genetic deletion of PARP1 as well as by pharmacological PARP inhibition. Moreover, impaired repair of hydrogen peroxide and CPT-induced DNA damage in XRCC1 KO cells could be partially rescued by additional deletion of PARP1. Our results therefore highlight important reciprocal regulatory functions of XRCC1 and PARP1 during genotoxic stress response.
Asunto(s)
Daño del ADN , NAD , Humanos , NAD/metabolismo , Reparación del ADN , Células HeLa , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismoRESUMEN
Energy generation in the brain to ameliorate energy deficit in migraine leads to oxidative stress as it is associated with reactive oxygen species (ROS) that may damage DNA and show a pronociceptive action in meninges mediated by transient receptor potential cation channel subfamily A member 1 (TRPA1). Recent studies show high levels of single-strand breaks (SSBs) at specific sites in the genome of postmitotic neurons and point at SSB repair (SSBR) as an important element of homeostasis of the central nervous system. DNA topoisomerase 1 (TOP1) is stabilized in the DNA damage-inducing state by neuronal stimulation, including cortical spreading depression. Impairment in poly (ADP-ribose) polymerase 1 (PARP-1) and X-ray repair cross complementing 1 (XRCC1), key SSBR proteins, may be linked with migraine by transient receptor potential melastatin 2 (TRPM2). TRPM2 may also mediate the involvement of migraine-related neuroinflammation with PARP-1 activated by oxidative stress-related SSBs. In conclusion, aberrant activity of SSBR evoked by compromised PARP-1 and XRCC1 may contribute to pathological phenomena in the migraine brain. Such aberrant SSBR results in the lack of repair or misrepair of SSBs induced by ROS or resulting from impaired TOP1. Therefore, components of SSBR may be considered a prospective druggable target in migraine.
Asunto(s)
Trastornos Migrañosos , Canales Catiónicos TRPM , Humanos , Reparación del ADN , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismo , Proteínas de Unión al ADN/metabolismo , Roturas del ADN de Cadena Simple , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Estudios Prospectivos , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Estrés Oxidativo , Daño del ADNRESUMEN
Single-stranded breaks (SSBs) are the most frequent DNA lesions threatening genomic integrity. A highly kinked DNA structure in complex with human PARP-1 domains led to the proposal that SSB sensing in Eukaryotes relies on dynamics of both the broken DNA double helix and PARP-1's multi-domain organization. Here, we directly probe this process at the single-molecule level. Quantitative smFRET and structural ensemble calculations reveal how PARP-1's N-terminal zinc fingers convert DNA SSBs from a largely unperturbed conformation, via an intermediate state into the highly kinked DNA conformation. Our data suggest an induced fit mechanism via a multi-domain assembly cascade that drives SSB sensing and stimulates an interplay with the scaffold protein XRCC1 orchestrating subsequent DNA repair events. Interestingly, a clinically used PARP-1 inhibitor Niraparib shifts the equilibrium towards the unkinked DNA conformation, whereas the inhibitor EB47 stabilizes the kinked state.