RESUMEN
Mammalian DNA replication relies on various DNA helicase and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN-FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks.
Asunto(s)
ADN Helicasas , Replicación del ADN , RecQ Helicasas , Helicasa del Síndrome de Werner , RecQ Helicasas/metabolismo , RecQ Helicasas/genética , Replicación del ADN/efectos de los fármacos , Humanos , Helicasa del Síndrome de Werner/metabolismo , Helicasa del Síndrome de Werner/genética , ADN Helicasas/metabolismo , ADN Helicasas/genética , Telómero/metabolismo , Telómero/genética , Endonucleasas de ADN Solapado/metabolismo , Endonucleasas de ADN Solapado/genética , Proteasas Ubiquitina-Específicas/metabolismo , Proteasas Ubiquitina-Específicas/genética , Células HeLa , Células HEK293 , Homeostasis del Telómero/efectos de los fármacos , Cromatina/metabolismoRESUMEN
Persistent DNA-protein crosslinks formed by human topoisomerase IIIα (TOP3A-DPCs) interfere with DNA metabolism and lead to genome damage and cell death. Recently, we demonstrated that such abortive TOP3A-DPCs are ubiquitylated and proteolyzed by Spartan (SPRTN). Here, we identify transient poly(ADP-ribosylation) (PARylation) in addition to ubiquitylation as a signaling mechanism for TOP3A-DPC repair and provide evidence that poly(ADP-ribose) polymerase 1 (PARP1) drives the repair of TOP3A-DPCs by recruiting flap endonuclease 1 (FEN1) to the TOP3A-DPCs. We find that blocking PARylation attenuates the interaction of FEN1 and TOP3A and that TOP3A-DPCs accumulate in cells with compromised PARP1 activity and in FEN1-deficient cells. We also show that PARP1 suppresses TOP3A-DPC ubiquitylation and that inhibiting the ubiquitin-activating enzyme E1 (UBE1) increases TOP3A-DPCs, consistent with ubiquitylation serving as a signaling mechanism for TOP3A-DPC repair mediated by SPRTN and TDP2. We propose that two concerted pathways repair TOP3A-DPCs: PARylation-driven FEN1 excision and ubiquitylation-driven SPRTN-TDP2 excision.
Asunto(s)
Reparación del ADN , ADN-Topoisomerasas de Tipo I , Endonucleasas de ADN Solapado , Poli(ADP-Ribosa) Polimerasa-1 , Ubiquitinación , Humanos , Endonucleasas de ADN Solapado/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Poli ADP Ribosilación , ADN/metabolismoRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium that causes a wide range of illnesses, necessitating the development of new technologies for its detection. Herein, we propose a graphene oxide (GO)-based sensing platform for the detection of mecA gene in MRSA using flap endonuclease 1 (FEN1)-assisted target recycling and Klenow fragment (KF)-triggered signal amplification. Without the target, all the DNA probes were adsorbed onto GO, resulting in fluorescence quenching of the dye. Upon the addition of the target, a triple complex was formed that triggered FEN1-assisted target recycling and initiated two polymerization reactions with the assistance of KF polymerase, generating numerous dsDNA that were repelled by GO. These dsDNAs triggered fluorescence enhancement when SYBR Green I was added. Therefore, the target DNA was quantified by measuring the fluorescence at excitation and emission wavelengths of 480/526 nm. This mecA gene assay showed a good linear range from 1 to 50 nM with a lower limit of detection of 0.26 nM, and displayed good applicability to the analysis of real samples. Thus, a new method for monitoring MRSA has been developed that has great potential for early clinical diagnosis and treatment.
Asunto(s)
Proteínas Bacterianas , Técnicas Biosensibles , Endonucleasas de ADN Solapado , Grafito , Staphylococcus aureus Resistente a Meticilina , Proteínas de Unión a las Penicilinas , Grafito/química , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Proteínas Bacterianas/genética , Proteínas de Unión a las Penicilinas/genética , Técnicas Biosensibles/métodos , Fluorometría/métodos , ADN Polimerasa I/genética , ADN Polimerasa I/metabolismo , ADN Bacteriano/genética , Límite de DetecciónRESUMEN
To achieve highly sensitive and reliable detection of apurinic/apyrimidinic endonuclease 1 (APE1), a critical cancer diagnostic biomarker, we designed a DNA walker-based dual-mode biosensor, utilizing cellular endogenous dual enzymes (APE 1 and Flap endonuclease 1 (FEN 1)) to collaborate in activating and propelling DNA walker motion on DNA-functionalized Au nanoparticles. Incorporating both fluorescence and electrochemical detection modes, this system leverages signal amplification from DNA walker movement and cascade amplification through tandem hybridization chain reactions (HCR), achieving highly sensitive detection of APE 1. In the fluorescence mode, continuous DNA walker movement, initiated by APE1 and driven by FEN1, generates a robust signal response within a concentration range of 0.01-500 U mL-1, presenting a good linearity in the concentration range of 0.01-10 U mL-1, with a detection limit of 0.01 U mL-1. In the electrochemical detection module, the cascade upstream DNA walker and downstream HCR dual signal amplification strategy further enhances the sensitivity of APE1 detection, extending the linear range to 0.01-50 U mL-1 and reducing the detection limit to 0.002 U mL-1. Rigorous validation demonstrates the biosensor's specificity and anti-interference capability against multiple enzymes. Moreover, it effectively distinguishes cancer cells from normal cell lysates, exhibiting excellent stability and consistency in the dual-modes. Overall, our findings underscore the efficacy of the developed dual-mode biosensor for detecting APE1 in serum and cell lysates samples, indicating its potential for clinical applications in disease diagnosis.
Asunto(s)
Técnicas Biosensibles , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN , Endonucleasas de ADN Solapado , Oro , Límite de Detección , Nanopartículas del Metal , Técnicas Biosensibles/métodos , Humanos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , ADN/química , Endonucleasas de ADN Solapado/química , Endonucleasas de ADN Solapado/metabolismo , Nanopartículas del Metal/química , Oro/química , Técnicas Electroquímicas/métodosRESUMEN
Nucleotide excision repair (NER) is the most universal repair pathway, which removes a wide range of DNA helix-distorting lesions caused by chemical or physical agents. The final steps of this repair process are gap-filling repair synthesis and subsequent ligation. XPA is the central NER scaffolding protein factor and can be involved in post-incision NER stages. Replication machinery is loaded after the first incision of the damaged strand that is performed by the XPF-ERCC1 nuclease forming a damaged 5'-flap processed by the XPG endonuclease. Flap endonuclease I (FEN1) is a critical component of replication machinery and is absolutely indispensable for the maturation of newly synthesized strands. FEN1 also contributes to the long-patch pathway of base excision repair. Here, we use a set of DNA substrates containing a fluorescently labeled 5'-flap and different size gap to analyze possible repair factor-replication factor interactions. Ternary XPA-FEN1-DNA complexes with each tested DNA are detected. Furthermore, we demonstrate XPA-FEN1 complex formation in the absence of DNA due to protein-protein interaction. Functional assays reveal that XPA moderately inhibits FEN1 catalytic activity. Using fluorescently labeled XPA, formation of ternary RPA-XPA-FEN1 complex, where XPA accommodates FEN1 and RPA contacts simultaneously, can be proposed. We discuss possible functional roles of the XPA-FEN1 interaction in NER related DNA resynthesis and/or other DNA metabolic processes where XPA can be involved in the complex with FEN1.
Asunto(s)
Reparación del ADN , Endonucleasas de ADN Solapado , Proteína de la Xerodermia Pigmentosa del Grupo A , Endonucleasas de ADN Solapado/metabolismo , Endonucleasas de ADN Solapado/genética , Humanos , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo A/genética , ADN/metabolismo , Unión Proteica , Reparación por EscisiónRESUMEN
Single-nucleotide polymorphism (SNP) is widely used in the study of disease-related genes and in the genetic study of animal and plant strains. Therefore, SNP detection is crucial for biomedical diagnosis and treatment as well as for molecular design breeding of animals and plants. In this regard, this article describes a novel technique for detecting SNP using flap endonuclease 1 (FEN 1) as a specific recognition element and catalytic hairpin assembly (CHA) cascade reaction as a signal amplification strategy. The mutant target (MT) was hybridized with a biotin-modified upstream probe and hairpin-type downstream probe (DP) to form a specific three-base overlapping structure. Then, FEN 1 was employed for three-base overlapping structure-specific recognition, namely, the precise SNP site identification and the 5' flap of DP dissociation. After dissociation, the hybridized probes were magnetically separated by a streptavidin-biotin complex. Especially, the ability to establish such a hairpin-type DP provided a powerful tool that could be used to hide the cut sequence (CS) and avoid false-positive signals. The cleaved CS initiated the CHA reaction and allowed superior fluorescence signal generation. Owing to the high specificity of FEN 1 for single base recognition, only the MT could be distinguished from the wild-type target and mismatched DNA. Owing to the dual signal amplification, as low as 0.36 fM MT and 1% mutation abundance from the mixtures could be detected, respectively. Furthermore, it could accurately identify SNPs from human cancer cells, as well as soybean leaf genome extracts. This strategy paves the way for the development of more precise and sensitive tools for diagnosing early onset diseases as well as molecular design breeding tools.
Asunto(s)
Endonucleasas de ADN Solapado , Polimorfismo de Nucleótido Simple , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , Hibridación de Ácido NucleicoRESUMEN
Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.
Asunto(s)
Cromatografía de Afinidad , Cromatografía de Afinidad/métodos , Endonucleasas de ADN Solapado/química , Endonucleasas de ADN Solapado/aislamiento & purificación , Endonucleasas de ADN Solapado/metabolismo , Cromatografía Liquida/métodos , Histidina/química , Escherichia coli/genética , Escherichia coli/química , Escherichia coli/metabolismo , Oligopéptidos/química , Oligopéptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
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
BACKGROUND: The metastasis accounts for most deaths from breast cancer (BRCA). Understanding the molecular mechanisms of BRCA metastasis is urgently demanded. Flap Endonuclease 1 (FEN1), a pivotal factor in DNA metabolic pathways, contributes to tumor growth and drug resistance, however, little is known about the role of FEN1 in BRCA metastasis. METHODS AND RESULTS: In this study, FEN1 expression and its clinical correlation in BRCA were investigated using bioinformatics, showing being upregulated in BRCA samples and significant relationships with tumor stage, node metastasis, and prognosis. Immunohistochemistry (IHC) staining of local BRCA cohort indicated that the ratio of high FEN1 expression in metastatic BRCA tissues rose over that in non-metastatic tissues. The assays of loss-of-function and gain-of-function showed that FEN1 enhanced BRCA cell proliferation, migration, invasion, xenograft growth as well as lung metastasis. It was further found that FEN1 promoted the aggressive behaviors of BRCA cells via Signal Transducer and Activator of Transcription 3 (STAT3) activation. Specifically, the STAT3 inhibitor Stattic thwarted the FEN1-induced enhancement of migration and invasion, while the activator IL-6 rescued the decreased migration and invasion caused by FEN1 knockdown. Additionally, overexpression of FEN1 rescued the inhibitory effect of nuclear factor-κB (NF-κB) inhibitor BAY117082 on phosphorylated STAT3. Simultaneously, the knockdown of FEN1 attenuated the phosphorylation of STAT3 promoted by the NF-κB activator tumor necrosis factor α (TNF-α). CONCLUSIONS: These results indicate a novel mechanism that NF-κB-driven FEN1 contributes to promoting BRCA growth and metastasis by STAT3 activation.
Asunto(s)
Neoplasias de la Mama , Endonucleasas de ADN Solapado , Factor de Transcripción STAT3 , Femenino , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Animales , RatonesRESUMEN
This study investigated the fabrication of gallic acid-loaded chitosan nanoparticles (Gal-Chi-NPs) that enhanced the DNA damage and apoptotic features by inhibiting FEN-1 expressions in MDA-MB 231 cells. Gal-Chi-NPs were fabricated by the ionic gelation method, and it was characterized by several studies such as dynamic light spectroscopy, Fourier-transforms infrared spectroscopy, x-ray diffraction, scanning electron microscopy, energy-dispersive x-ray, atomic force microscopy, and thermogravimetric analysis. We have obtained that Gal-Chi-NPs displayed 182.2 nm with crystal, smooth surface, and heat stability in nature. Gal-Chi-NPs induce significant toxicity in MDA-MB-231 cells that compared with normal NIH-3T3 cells. A significant reactive oxygen species (ROS) overproduction was observed in Gal-Chi-NPs treated MDA-MB-231. Flap endonuclease-1 (FEN-1) is a crucial protein involved in long patch base excision repair that is involved in repairing the chemotherapeutic mediated DNA-damaged base. Therefore, inhibition of FEN-1 protein expression is a crucial target for enhancing chemotherapeutical efficacy. In this study, we have obtained that Gal-Chi-NPs treatment enhanced the DNA damage by observing increased p-H2AX, PARP1; and suppressed the expression of FEN-1 in MDA-MB-231 cells. Moreover, Gal-Chi-NPs inhibited the expression of tumor proliferating markers p-PI3K, AKT, cyclin-D1, PCNA, and BCL-2; induced proapoptotic proteins (Bax and caspase-3) in MDA-MB 231 cells. Thus, Gal-Chi-NPs induce DNA damage and apoptotic features and inhibit tumor proliferation by suppressing FEN-1 expression in triple-negative breast cancer cells.
Asunto(s)
Apoptosis , Quitosano , Daño del ADN , Endonucleasas de ADN Solapado , Ácido Gálico , Nanopartículas , Neoplasias de la Mama Triple Negativas , Ácido Gálico/farmacología , Ácido Gálico/química , Quitosano/química , Humanos , Endonucleasas de ADN Solapado/metabolismo , Daño del ADN/efectos de los fármacos , Apoptosis/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Nanopartículas/química , Nanopartículas/toxicidad , Línea Celular Tumoral , Ratones , Animales , Especies Reactivas de Oxígeno/metabolismo , Femenino , Células 3T3 NIHRESUMEN
The overexpression of Flap endonuclease 1 (FEN1) has been implicated in drug resistance and prognosis across various cancer types. However, the precise role of FEN1 in colon cancer remains to be fully elucidated. In this study, we employed comprehensive datasets from The Cancer Genome Atlas, Gene Expression Omnibus, and Human Protein Atlas to examine FEN1 expression and assess its correlation with clinical pathology and prognosis in colon cancer. We utilized the pRRophetic algorithm to evaluate drug sensitivity and performed differential expression analysis to identify genes associated with FEN1-mediated drug sensitivity. Gene set enrichment analysis was conducted to further investigate these genes. Additionally, single-cell sequencing analysis was employed to explore the relationship between FEN1 expression and functional states. Cox regression analysis was implemented to construct a prognostic model, and a nomogram for prognosis was developed. Our analysis of The Cancer Genome Atlas and Gene Expression Omnibus datasets revealed a significant upregulation of FEN1 in colon cancer. However, while FEN1 expression showed no notable correlation with prognosis, it displayed associations with metastasis. Single-cell sequencing analysis further confirmed a positive correlation between FEN1 expression and colon cancer metastasis. Furthermore, we detected marked discrepancies in drug responsiveness between the High_FEN1 and Low_FEN1 groups, identifying 342 differentially expressed genes. Enrichment analysis showed significant suppression in processes related to DNA replication, spliceosome, and cell cycle pathways in the Low_FEN1 group, while the calcium signaling pathway, cAMP signaling pathway, and other pathways were activated. Of the 197 genes differentially expressed and strongly linked to FEN1 expression, 39 were significantly implicated in colon cancer prognosis. Finally, we constructed a risk signature consisting of 5 genes, which, when combined with drug treatment and pathological staging, significantly improved the prediction of colon cancer prognosis. This study offers novel insights into the interplay among FEN1 expression levels, colon cancer metastatic potential, and sensitivity to therapeutic agents. Furthermore, we successfully developed a multi-gene prognostic risk signature derived from FEN1.
Asunto(s)
Neoplasias del Colon , Endonucleasas de ADN Solapado , Humanos , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Pronóstico , Resistencia a Medicamentos , Biología ComputacionalRESUMEN
To improve breast cancer treatment and to enable new strategies for therapeutic resistance, therapeutic targets are constantly being studied. Potential targets are proteins of DNA repair and replication and genomic integrity, such as Flap Endonuclease 1 (FEN1). This study investigated the effects of FEN1 inhibitor FEN1-IN-4 in combination with ionizing radiation on cell death, clonogenic survival, the cell cycle, senescence, doubling time, DNA double-strand breaks and micronuclei in breast cancer cells, breast cells and healthy skin fibroblasts. Furthermore, the variation in the baseline FEN1 level and its influence on treatment prognosis was investigated. The cell lines show specific response patterns in the aspects studied and have heterogeneous baseline FEN1 levels. FEN1-IN-4 has cytotoxic, cytostatic and radiosensitizing effects, expressed through increasing cell death by apoptosis and necrosis, G2M share, senescence, double-strand breaks and a reduced survival fraction. Nevertheless, some cells are less affected by the cytotoxicity and fibroblasts show a rather limited response. In vivo, high FEN1 mRNA expression worsens the prognosis of breast cancer patients. Due to the increased expression in breast cancer tissue, FEN1 could represent a new tumor and prognosis marker and FEN1-IN-4 may serve as a new potent agent in personalized medicine and targeted breast cancer therapy.
Asunto(s)
Antineoplásicos , Neoplasias de la Mama , Endonucleasas de ADN Solapado , Femenino , Humanos , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Reparación del ADN , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , PronósticoRESUMEN
Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.
Asunto(s)
Neoplasias , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Reparación del ADN , Daño del ADN , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Endonucleasas de ADN Solapado/uso terapéutico , Exodesoxirribonucleasas/genética , Enzimas Reparadoras del ADN/genéticaRESUMEN
PURPOSE: Cholangiocarcinoma (CHOL) comprises a cluster of highly heterogeneous malignant biliary tumors. Flap endonuclease-1 (FEN1) is a member of the Rad2 structure-specific nuclease family. This study aimed to explore the biological functions and mechanisms of FEN1 in CHOL. METHODS: FEN1 expression was analyzed in tissues of patients with CHOL and FEN1 mutations. We observe the influence of FEN1 on cellular proliferation, migration, and invasion, as well as on DNA damage repair and glycolysis. Western blotting was performed to determine the regulatory mechanism of FEN1 in CHOL progression. RESULTS: FEN1 was highly expressed in the cancer tissues of CHOL patients. The high mutation rate of FEN1 in CHOL tissues was mainly due to the amplified repeats. FEN1 promotes the proliferation, migration, and invasion of HUCCT1 and QBC939 cells. In addition, FEN1 induced DNA damage repair and aerobic glycolysis in CHOL cells. FEN1 also promoted xenograft tumor growth in vivo. Moreover, we showed that FEN1 mediated the epithelial-mesenchymal transition (EMT) of CHOL. FEN1-mediated EMT was found to be transduced by the Wnt/ß-catenin signaling pathway. CONCLUSION: FEN1 was significantly overexpressed in CHOL tissues, and FEN1 regulates the progression of CHOL through the Wnt/ß-catenin signaling pathway.
Asunto(s)
Neoplasias de los Conductos Biliares , Colangiocarcinoma , Humanos , Vía de Señalización Wnt/genética , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Línea Celular Tumoral , Colangiocarcinoma/genética , Neoplasias de los Conductos Biliares/genética , Conductos Biliares Intrahepáticos , Transición Epitelial-Mesenquimal/genética , Proliferación Celular/genética , beta Catenina/genética , beta Catenina/metabolismo , Regulación Neoplásica de la Expresión Génica , Movimiento CelularRESUMEN
It is well known that chimeric antigen receptor T-cell immunotherapy (CAR-T-cell immunotherapy) has excellent therapeutic effect in haematological tumours, but it still faces great challenges in solid tumours, including inefficient T-cell tumour infiltration and poor functional persistence. Flap structure-specific endonuclease 1 (FEN1), highly expressed in a variety of cancer cells, plays an important role in both DNA replication and repair. Previous studies have reported that FEN1 inhibition is an effective strategy for cancer treatment. Therefore, we hypothesized whether FEN1 inhibitors combined with CAR-T-cell immunotherapy would have a stronger killing effect on solid tumours. The results showed that low dose of FEN1 inhibitors SC13 could induce an increase of double-stranded broken DNA (dsDNA) in the cytoplasm. Cytosolic dsDNA can activate the cyclic GMP-AMP synthase-stimulator of interferon gene signalling pathway and increase the secretion of chemokines. In vivo, under the action of FEN1 inhibitor SC13, more chemokines were produced at solid tumour sites, which promoted the infiltration of CAR-T cells and improved anti-tumour immunity. These findings suggest that FEN1 inhibitors could enable CAR-T cells to overcome poor T-cell infiltration and improve the treatment of solid tumours.
Asunto(s)
Neoplasias , Humanos , Transducción de Señal , ADN , Linfocitos T/metabolismo , Nucleotidiltransferasas/genética , Quimiocinas , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismoRESUMEN
PURPOSE: Flap endonuclease 1 (FEN1) is highly upregulated in prostate cancer and promotes the growth of prostate cancer cells. Androgen receptor (AR) is the most critical determinant of the occurrence, progression, metastasis, and treatment of prostate cancer. However, the effect of FEN1 on docetaxel (DTX) sensitivity and the regulatory mechanisms of AR on FEN1 expression in prostate cancer need to be further studied. METHODS: Bioinformatics analyses were performed using data from the Cancer Genome Atlas and the Gene Expression Omnibus. Prostate cancer cell lines 22Rv1 and LNCaP were used. FEN1 siRNA, FEN1 overexpression plasmid, and AR siRNA were transfected into cells. Biomarker expression was evaluated by immunohistochemistry and Western blotting. Apoptosis and the cell cycle were explored using flow cytometry analysis. Luciferase reporter assay was performed to verify the target relationship. Xenograft assays were conducted using 22Rv1 cells to evaluate the in vivo conclusions. RESULTS: Overexpression of FEN1 inhibited cell apoptosis and cell cycle arrest in the S phase induced by DTX. AR knockdown enhanced DTX-induced cell apoptosis and cell cycle arrest at the S phase in prostate cancer cells, which was attenuated by FEN1 overexpression. In vivo experiments showed that overexpression of FEN1 significantly increased tumour growth and weakened the inhibitory effect of DTX on prostate tumour growth, while AR knockdown enhance the sensitivity of DTX to prostate tumour. AR knockdown resulted in FEN1, pho-ERK1/2, and pho-ELK1 downregulation, and the luciferase reporter assay confirmed that ELK1 can regulate the transcription of FEN1. CONCLUSION: Collectively, our studies demonstrate that AR knockdown improves the DTX sensitivity of prostate cancer cells by downregulating FEN1 through the ERK/ELK1 signalling pathway.
Asunto(s)
Neoplasias de la Próstata , Receptores Androgénicos , Masculino , Humanos , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Sistema de Señalización de MAP Quinasas , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Proliferación Celular , Línea Celular Tumoral , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Docetaxel/farmacología , ARN Interferente Pequeño/metabolismo , Proteína Elk-1 con Dominio ets/genética , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
Oral squamous cell carcinoma (OSCC) escape from the immune system is mediated through several immunosuppressive phenotypes that are critical to the initiation and progression of tumors. As a hallmark of cancer, DNA damage repair is closely related to changes in the immunophenotypes of tumor cells. Although flap endonuclease-1 (FEN1), a pivotal DNA-related enzyme is involved in DNA base excision repair to maintain the stability of the cell genome, the correlation between FEN1 and tumor immunity has been unexplored. In the current study, by analyzing the clinicopathological characteristics of FEN1, we demonstrated that FEN1 overexpressed and that an inhibitory immune microenvironment was established in OSCC. In addition, we found that downregulating FEN1 inhibited the growth of OSCC tumors. In vitro studies provided evidence that FEN1 knockdown inhibited the biological behaviors of OSCC and caused DNA damage. Performing multiplex immunohistochemistry (mIHC), we directly observed that the acquisition of critical immunosuppressive phenotypes was correlated with the expression of FEN1. More importantly, FEN1 directly or indirectly regulated two typical immunosuppressive phenotype-related proteins human leukocyte antigen (HLA-DR) and programmed death receptor ligand 1 (PD-L1), through the interferon-gamma (IFN-γ)/janus kinase (JAK)/signal transducer and activator transcription 1 (STAT1) pathway. Our study highlights a new perspective on FEN1 action for the first time, providing theoretical evidence that it may be a potential immunotherapy target for OSCC.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias de la Boca , Humanos , Carcinoma de Células Escamosas/patología , ADN , Regulación hacia Abajo , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Interferón gamma/farmacología , Interferón gamma/metabolismo , Neoplasias de la Boca/patología , Fenotipo , Carcinoma de Células Escamosas de Cabeza y Cuello , Microambiente Tumoral , Quinasas Janus/metabolismoRESUMEN
The structure-specific endonuclease flap endonuclease 1 (FEN1) is an essential functional protein in DNA replication and genome stability, and it has been identified as a promising biomarker and drug target for multiple cancers. Herein, we develop a target-activated T7 transcription circuit-mediated multiple cycling signal amplification platform for monitoring FEN1 activity in cancer cells. In the presence of FEN1, the flapped dumbbell probe is cleaved to generate a free 5' flap single-stranded DNA (ssDNA) with the 3'-OH terminus. The ssDNA can hybridize with the T7 promoter-bearing template probe to trigger the extension with the aid of Klenow fragment (KF) DNA polymerase. Upon the addition of T7 RNA polymerase, an efficient T7 transcription amplification reaction is initiated to produce abundant single-stranded RNAs (ssRNAs). The ssRNA can hybridize with a molecular beacon to form an RNA/DNA heteroduplex that can be selectively digested by DSN to generate an enhanced fluorescence signal. This method exhibits good specificity and high sensitivity with a limit of detection (LOD) of 1.75 × 10-6 U µL-1. Moreover, it can be applied for the screening of FEN1 inhibitors and the monitoring of FEN1 activity in human cells, holding great potential in drug discovery and clinical diagnosis.
Asunto(s)
Endonucleasas de ADN Solapado , Neoplasias , Humanos , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , ADN/genética , ADN/metabolismo , Replicación del ADN , Reparación del ADN , Neoplasias/genéticaRESUMEN
The current model for Okazaki fragment maturation in bacteria invokes RNA cleavage by RNase H, followed by strand displacement synthesis and 5' RNA flap removal by DNA polymerase I (Pol I). RNA removal by Pol I is thought to occur through the 5'-3' flap endo/exonuclease (FEN) domain, located in the N-terminus of the protein. In addition to Pol I, many bacteria encode a second, Pol I-independent FEN. The contribution of Pol I and Pol I-independent FENs to DNA replication and genome stability remains unclear. In this work we purified Bacillus subtilis Pol I and FEN, then assayed these proteins on a variety of RNA-DNA hybrid and DNA-only substrates. We found that FEN is far more active than Pol I on nicked double-flap, 5' single flap, and nicked RNA-DNA hybrid substrates. We show that the 5' nuclease activity of B. subtilis Pol I is feeble, even during DNA synthesis when a 5' flapped substrate is formed modeling an Okazaki fragment intermediate. Examination of Pol I and FEN on DNA-only substrates shows that FEN is more active than Pol I on most substrates tested. Further experiments show that ΔpolA phenotypes are completely rescued by expressing the C-terminal polymerase domain while expression of the N-terminal 5' nuclease domain fails to complement ΔpolA. Cells lacking FEN (ΔfenA) show a phenotype in conjunction with an RNase HIII defect, providing genetic evidence for the involvement of FEN in Okazaki fragment processing. With these results, we propose a model where cells remove RNA primers using FEN while upstream Okazaki fragments are extended through synthesis by Pol I. Our model resembles Okazaki fragment processing in eukaryotes, where Pol δ catalyzes strand displacement synthesis followed by 5' flap cleavage using FEN-1. Together our work highlights the conservation of ordered steps for Okazaki fragment processing in cells ranging from bacteria to human.
Asunto(s)
Bacillus subtilis , Endonucleasas de ADN Solapado , Humanos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , ADN/genética , Replicación del ADN/genética , ARN/metabolismo , Exonucleasas/genéticaRESUMEN
BACKGROUND: Flap endonuclease 1 (FEN1), well known for its structural-specific nuclease, possessing 5'-flap endonuclease and 5'-3' exonuclease activities, is mainly involved in DNA replication and repair. Protein lysine acetylation is an important posttranslational modification that could regulate numerous proteins' activity, subcellular localization, protein-protein interaction etc., and influences many biological processes. Our previous studies on integrated succinylome profiles found that succinylation and acetylation levels of FEN1 would change under different conditions. Succinylation at FEN1 Lys200 site results in the accumulation of damaged DNA and increased susceptibility to fork-stalling agents. The interplay with other forms of modification could affects its protein interaction affinity and thus contribute to genome stability. OBJECTIVE: This article studied the biological role of FEN1 by acyl modification in HeLa cells. METHOD: In order to explore the function of FEN1 acylation in cells, we mimicked the presence or absence of acetylation or succinylation by mutating key amino acids to glutamic acid and glutamine. We carried out a series of experiments including cell cycle, MTS, enzyme kinetics measurements, immunofluorescence and so on. RESULTS: The absence of acylation of FEN1 leads to the blocked cell cycle process and the reduced efficiency of FEN1 on its DNA substrates, affecting the interaction of FEN1 with both repair and replication related proteins and thus its role in the repair of DNA damage. CONCLUSION: We have verified acyl groups could modify Lys125, Lys252 and Lys254 of FEN1. Acylation level of these three is important for enzyme activity, cell proliferation and DNA damage response, thus contributing to genome stability.