RESUMEN
Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a recessively inherited neurodegenerative disorder caused by intronic biallelic, nonreference CCCTT/AAGGG repeat expansions within RFC1. To investigate how these repeats cause disease, we generated patient induced pluripotent stem cell-derived neurons (iNeurons). CCCTT/AAGGG repeat expansions do not alter neuronal RFC1 splicing, expression, or DNA repair pathway function. In reporter assays, AAGGG repeats are translated into pentapeptide repeat proteins. However, these proteins and repeat RNA foci were not detected in iNeurons, and overexpression of these repeats failed to induce neuronal toxicity. CANVAS iNeurons exhibit defects in neuronal development and diminished synaptic connectivity that is rescued by CRISPR deletion of a single expanded AAGGG allele. These deficits were neither replicated by RFC1 knockdown in control iNeurons nor rescued by RFC1 reprovision in CANVAS iNeurons. These findings support a repeat-dependent but RFC1 protein-independent cause of neuronal dysfunction in CANVAS, with implications for therapeutic development in this currently untreatable condition.
Asunto(s)
Ataxia Cerebelosa , Expansión de las Repeticiones de ADN , Células Madre Pluripotentes Inducidas , Neuronas , Proteína de Replicación C , Sinapsis , Humanos , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Neuronas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Expansión de las Repeticiones de ADN/genética , Ataxia Cerebelosa/genética , Ataxia Cerebelosa/patología , Ataxia Cerebelosa/metabolismo , Sinapsis/metabolismo , Sinapsis/genética , Vestibulopatía Bilateral/genética , Vestibulopatía Bilateral/metabolismo , Enfermedades Vestibulares/genética , AlelosRESUMEN
BACKGROUND: Colorectal cancer (CRC) remains a global health concern with persistently high incidence and mortality rates. However, the specific pathogenesis of CRC remains poorly understood. This study aims to investigate the role and pathogenesis of serine and arginine rich splicing factor 10 (SRSF10) in colorectal cancer. METHODS: Bioinformatics analysis was employed to predict SRSF10 gene expression in CRC patients. Functional experiments involving SRSF10 knockdown and overexpression were conducted using CCK8, transwell, scratch assay, and flow cytometry. Additionally, the PRIdictor website was utilized to predict the SRSF10 interaction site with RFC5. The identification of different transcripts of SRSF10-acting RFC5 pre-mRNA was achieved through agarose gel electrophoresis. RESULT: The knockdown of SRSF10 inhibited the proliferation and migration ability of CRC cells, while promoting apoptosis and altering the DNA replication of CRC cells. Conversely, when SRSF10 was highly expressed, it enhanced the proliferation and migration ability of CRC cells and caused changes in the cell cycle of colorectal cancer cells. This study revealed a change in the replicating factor C subunit 5 (RFC5) gene in colorectal cancer cells following SRSF10 knockdown. Furthermore, it was confirmed that SRSF10 increased RFC5 exon2-AS1(S) transcription variants, thereby promoting the development of colorectal cancer through AS1 exclusion to exon 2 of RFC5. CONCLUSION: In summary, this study demonstrates that SRSF10 promotes the progression of colorectal cancer by generating an aberrantly spliced exclusion isoform of AS1 within RFC5 exon 2. These findings suggest that SRSF10 could serve as a crucial target for the clinical diagnosis and treatment of CRC.
Asunto(s)
Empalme Alternativo , Apoptosis , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Proteína de Replicación C , Factores de Empalme Serina-Arginina , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo , Proliferación Celular/genética , Movimiento Celular/genética , Apoptosis/genética , Línea Celular Tumoral , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas Represoras , Proteínas de Ciclo CelularRESUMEN
The proliferating cell nuclear antigen (PCNA) clamp encircles DNA to hold DNA polymerases (Pols) to DNA for processivity. The Ctf18-RFC PCNA loader, a replication factor C (RFC) variant, is specific to the leading-strand Pol (Polε). We reveal here the underlying mechanism of Ctf18-RFC specificity to Polε using cryo-electron microscopy and biochemical studies. We found that both Ctf18-RFC and Polε contain specific structural features that direct PCNA loading onto DNA. Unlike other clamp loaders, Ctf18-RFC has a disordered ATPase associated with a diverse cellular activities (AAA+) motor that requires Polε to bind and stabilize it for efficient PCNA loading. In addition, Ctf18-RFC can pry prebound Polε off of DNA, then load PCNA onto DNA and transfer the PCNA-DNA back to Polε. These elements in both Ctf18-RFC and Polε provide specificity in loading PCNA onto DNA for Polε.
Asunto(s)
Replicación del ADN , Antígeno Nuclear de Célula en Proliferación , Proteína de Replicación C , Humanos , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/química , Microscopía por Crioelectrón , ADN/química , ADN/metabolismo , ADN Polimerasa II/metabolismo , ADN Polimerasa II/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas Nucleares , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/química , Unión Proteica , Proteína de Replicación C/metabolismo , Proteína de Replicación C/química , Dominios ProteicosRESUMEN
The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease.
Asunto(s)
Proteína de Replicación C , Humanos , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Masculino , Células HeLa , Femenino , Fenotipo , Replicación del ADN/genética , Adulto , Mutación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , AlelosRESUMEN
RFC4 is required for DNA polymerase δ and DNA polymerase ε to initiate DNA template expansion. Downregulated RFC4 inhibits tumour proliferation by causing S-phase arrest and inhibiting mitosis, resulting in the reduction of tumour cells. RFC4 has been implicated that it plays an important role in the initiation and progression of cancers, but a comprehensive analysis of the role of RFC4 in cancer has not been performed. We comprehensively analysed the expression, prognosis, methylation level, splicing level, relationship of RFC4 and immune infiltration, and pan-cancer immunotherapy response used various databases (including TCGA, GTEx, UALCAN, Oncosplicing, TIDE, TISCH, HPA and CAMOIP), and experimented its biological function in HCC. Through pan-cancer analysis, we found that RFC4 is significantly upregulated in most tumours. The tumour patients with high expression of RFC4 have poor prognosis. The methylation level and variable splicing level of RFC4 were abnormal in most tumours compared with the adjacent tissues. Furthermore, RFC4 was closely associated with immune cell infiltration in various cancers. RFC4 was significantly co-expressed with immune checkpoints and other immune-related genes. The expression of RFC4 could indicate the immunotherapy efficacy of some tumours. The RFC4 expression was associated with sensitivity to specific small molecule drugs. Cell experiments have shown that downregulated RFC4 can inhibit cell cycle and tumour cell proliferation. We conducted a systematic pan-cancer analysis of RFC4, and the results showed that RFC4 can serve as a biomarker for cancer diagnosis and prognosis. These findings open new perspectives for precision medicine.
Asunto(s)
Biomarcadores de Tumor , Regulación Neoplásica de la Expresión Génica , Neoplasias , Proteína de Replicación C , Microambiente Tumoral , Humanos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Microambiente Tumoral/inmunología , Pronóstico , Proteína de Replicación C/metabolismo , Proteína de Replicación C/genética , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Línea Celular Tumoral , Metilación de ADN , Proliferación Celular , Inmunoterapia/métodosRESUMEN
DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, PCNA, carry out DNA synthesis during lagging strand replication, initiation of leading strand replication, and the major DNA damage repair and tolerance pathways. Pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process involving the major single strand DNA (ssDNA)-binding protein complex, RPA, the processivity sliding clamp loader, RFC, PCNA and pol δ. During this process, the interactions of RPA, RFC and pol δ with a P/T junction all significantly overlap. A burning issue that has yet to be resolved is how these overlapping interactions are accommodated during this process. To address this, we design and utilize novel, ensemble FRET assays that continuously monitor the interactions of RPA, RFC, PCNA and pol δ with DNA as pol δ holoenzymes are assembled and initiate DNA synthesis. Results from the present study reveal that RPA remains engaged with P/T junctions throughout this process and the RPAâ¢DNA complexes dynamically re-organize to allow successive binding of RFC and pol δ. These results have broad implications as they highlight and distinguish the functional consequences of dynamic RPAâ¢DNA interactions in RPA-dependent DNA metabolic processes.
Asunto(s)
ADN Polimerasa III , Replicación del ADN , ADN , Antígeno Nuclear de Célula en Proliferación , Proteína de Replicación A , Proteína de Replicación C , Moldes Genéticos , Proteína de Replicación A/metabolismo , ADN Polimerasa III/metabolismo , ADN Polimerasa III/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Holoenzimas/metabolismo , ADN/metabolismo , ADN/biosíntesis , Proteína de Replicación C/metabolismo , Proteína de Replicación C/genética , Cartilla de ADN/genética , Transferencia Resonante de Energía de Fluorescencia , HumanosRESUMEN
The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well-studied pentameric ATPase complex RFC (RFC1-5). The CTF18-RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo-EM analysis of in vitro assembled human CTF18-RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3'-ss/dsDNA junction by CTF18-RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique ß-hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3'-end of the primer; this ensures its ability to load PCNA to any 3'-ss/dsDNA junction, facilitated by the binding energy of the E-plug to the major groove. Our study reveals unique structural features of the human CTF18-RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas , Microscopía por Crioelectrón , Proteínas Nucleares , Antígeno Nuclear de Célula en Proliferación , Proteína de Replicación C , Humanos , Microscopía por Crioelectrón/métodos , ADN/metabolismo , ADN/química , Replicación del ADN , Modelos Moleculares , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/química , Unión Proteica , Proteína de Replicación C/metabolismo , Proteína de Replicación C/químicaRESUMEN
Short tandem repeats are inherently unstable during DNA replication depending on repeat length, and the expansion of the repeat length in the human genome is responsible for repeat expansion disorders. Pentanucleotide AAGGG and ACAGG repeat expansions in intron 2 of the gene encoding replication factor C subunit 1 (RFC1) cause cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and other phenotypes of late-onset cerebellar ataxia. Herein, we reveal the structural polymorphism of the RFC1 repeats associated with CANVAS in vitro. Single-stranded AAGGG repeat DNA formed a hybrid-type G-quadruplex, whereas its RNA formed a parallel-type G-quadruplex with three layers. The RNA of the ACAGG repeat formed hairpin structure comprising C-G and G-C base pairs with A:A and GA:AG mismatched repeats. Furthermore, both pathogenic repeat RNAs formed more rigid structures than those of the nonpathogenic repeat RNAs. These findings provide novel insights into the structural polymorphism of the RFC1 repeats, which may be closely related to the disease mechanism of CANVAS.
Asunto(s)
Ataxia Cerebelosa , Expansión de las Repeticiones de ADN , Enfermedades del Sistema Nervioso Periférico , Proteína de Replicación C , Enfermedades Vestibulares , Humanos , Ataxia Cerebelosa/genética , Ataxia Cerebelosa/metabolismo , G-Cuádruplex , Repeticiones de Microsatélite , Polimorfismo Genético , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Proteína de Replicación C/química , ARN/química , ARN/genética , ARN/metabolismo , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades Vestibulares/genética , Enfermedades Vestibulares/metabolismoRESUMEN
Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of the ring-shaped sliding clamp and the subsequent binding to primer-template (p/t)-junctions. The general architecture of clamp loaders is conserved across all life, suggesting that their mechanism is retained. Recent structural studies of the eukaryotic clamp loader replication factor C (RFC) revealed that it functions using a crab-claw mechanism, where clamp opening is coupled to a massive conformational change in the loader. Here we investigate the clamp loading mechanism of the Escherichia coli clamp loader at high resolution using cryo-electron microscopy. We find that the E. coli clamp loader opens the clamp using a crab-claw motion at a single pivot point, whereas the eukaryotic RFC loader uses motions distributed across the complex. Furthermore, we find clamp opening occurs in multiple steps, starting with a partly open state with a spiral conformation, and proceeding to a wide open clamp in a surprising planar geometry. Finally, our structures in the presence of p/t-junctions illustrate how the clamp closes around p/t-junctions and how the clamp loader initiates release from the loaded clamp. Our results reveal mechanistic distinctions in a macromolecular machine that is conserved across all domains of life.
Asunto(s)
Replicación del ADN , Escherichia coli , Microscopía por Crioelectrón , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Conformación Proteica , Proteína de Replicación C/metabolismo , Proteína de Replicación C/química , Proteína de Replicación C/genética , Modelos Moleculares , Estructura Cuaternaria de ProteínaRESUMEN
Colorectal cancer (CRC) ranks among the most prevalent cancers globally, with its incidence closely tied to DNA damage. The Replication Factor C (RFC) complexes comprises five protein subunits: RFC1, RFC2, RFC3, RFC4, and RFC5. These RFC complexes play crucial roles in DNA replication, repair pathways, activities post DNA damage, and ATP-dependent processes during DNA synthesis. However, the impact of RFC complexes proteins on CRC prognosis remains unclear. To explore this, we employed a computational analysis approach, utilizing platforms such as the DepMap portal, GEPIA, DAVID Bioinformatics for KEGG pathway analysis, Human Protein Atlas (HPA), STRING, and TIMER. Our results indicate that the mRNA levels of RFC1 and RFC5 were the least expressed among CRC cell lines compared to other RFC complex subunits. Notably, low RFC1 and RFC5 expression was correlated with poor prognosis in terms of CRC patients' overall survival (OS). Immunohistochemical results from the Human Protein Atlas demonstrated medium staining for RFC1, RFC2, and RFC5 in CRC tissues. Furthermore, the low expression of RFC1 and RFC5 showed a significant correlation with high expression levels of miR-26a-5p and miR-636, impacting cell proliferation through mismatch repair, DNA replication, and the nucleotide excision repair pathway. Although the precise functions of RFC1 in cancer are still unknown, our findings suggest that the small-molecule single target, CHEMBL430483, and multiple target molecules could be potential treatments for CRC. In conclusion, the elevated expression of miR-26a-5p and miR-636 targeting RFC1 and RFC5 expression holds promise as a potential biomarker for early-stage CRC detection. These insights provide novel directions and strategies for CRC therapies.
Asunto(s)
Neoplasias Colorrectales , MicroARNs , Proteínas de Saccharomyces cerevisiae , Humanos , Proteínas de Unión al ADN/genética , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Pronóstico , Neoplasias Colorrectales/genéticaRESUMEN
During DNA replication, the proliferating cell nuclear antigen (PCNA) clamps are loaded onto primed sites for each Okazaki fragment synthesis by the AAA+ heteropentamer replication factor C (RFC). PCNA encircling duplex DNA is quite stable and is removed from DNA by the dedicated clamp unloader Elg1-RFC. Here, we show the cryo-EM structure of Elg1-RFC in various states with PCNA. The structures reveal essential features of Elg1-RFC that explain how it is dedicated to PCNA unloading. Specifically, Elg1 contains two external loops that block opening of the Elg1-RFC complex for DNA binding, and an "Elg1 plug" domain that fills the central DNA binding chamber, thereby reinforcing the exclusive PCNA unloading activity of Elg1-RFC. Elg1-RFC was capable of unloading PCNA using non-hydrolyzable AMP-PNP. Both RFC and Elg1-RFC could remove PCNA from covalently closed circular DNA, indicating that PCNA unloading occurs by a mechanism that is distinct from PCNA loading. Implications for the PCNA unloading mechanism are discussed.
Asunto(s)
Proteínas de Unión al ADN , Proteínas de Saccharomyces cerevisiae , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/química , Proteína de Replicación C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
CANVAS is a recently characterized repeat expansion disease, most commonly caused by homozygous expansions of an intronic (A2G3)n repeat in the RFC1 gene. There are a multitude of repeat motifs found in the human population at this locus, some of which are pathogenic and others benign. In this study, we conducted structure-functional analyses of the pathogenic (A2G3)n and nonpathogenic (A4G)n repeats. We found that the pathogenic, but not the nonpathogenic, repeat presents a potent, orientation-dependent impediment to DNA polymerization in vitro. The pattern of the polymerization blockage is consistent with triplex or quadruplex formation in the presence of magnesium or potassium ions, respectively. Chemical probing of both repeats in vitro reveals triplex H-DNA formation by only the pathogenic repeat. Consistently, bioinformatic analysis of S1-END-seq data from human cell lines shows preferential H-DNA formation genome-wide by (A2G3)n motifs over (A4G)n motifs. Finally, the pathogenic, but not the nonpathogenic, repeat stalls replication fork progression in yeast and human cells. We hypothesize that the CANVAS-causing (A2G3)n repeat represents a challenge to genome stability by folding into alternative DNA structures that stall DNA replication.
Asunto(s)
Ataxia Cerebelosa , Expansión de las Repeticiones de ADN , Replicación del ADN , Enfermedades del Sistema Nervioso Periférico , Enfermedades Vestibulares , Humanos , ADN/metabolismo , ADN/química , ADN/genética , Expansión de las Repeticiones de ADN/genética , Replicación del ADN/genética , Conformación de Ácido Nucleico , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Ataxia Cerebelosa/genética , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades Vestibulares/genéticaRESUMEN
BACKGROUND: Gastric cancer (GC) is a malignant tumor with a high mortality rate, and thus, it is necessary to explore molecular mechanisms underlying its progression. While replication factor C subunit 3 (RFC3) has been demonstrated to function as an oncogene in many cancers, its role in GC remains unclear. METHODS: Tumor tissues were collected from clinical GC patients, and the expression of RFC3 was analyzed. NCI-N87 and HGC-27 cells were infected with lentivirus sh-RFC3 to knock down RFC3 expression. RFC3 expression levels were determined, in addition to cell biological behaviors both in vitro and in vivo. The relationship between RFC3 and the YAP1/TEAD signaling pathway was detected by dual luciferase reporter assay. RESULTS: RFC3 was upregulated in GC tumor tissues. RFC3 knockdown inhibited cell proliferation, promoted cell apoptosis of GC cells, and suppressed cell migration and invasion. Moreover, depleted RFC3 suppressed tumor growth and metastasis in vivo. Mechanistically, the YAP1/TEAD axis activated RFC3 expression transcriptionally by binding to the RFC3 promoter. CONCLUSIONS: RFC3 was transcriptional activated by the YAP1/TEAD signaling pathway, thus promoting GC progression. RFC3 may be a promising therapeutic target for GC.
Asunto(s)
Neoplasias Gástricas , Humanos , Neoplasias Gástricas/patología , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Línea Celular Tumoral , Transducción de Señal/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
Proliferating cell nuclear antigen (PCNA) is a homo-trimeric clamp complex that serves as the molecular hub for various DNA transactions, including DNA synthesis and post-replicative mismatch repair. Its timely loading and unloading are critical for genome stability. PCNA loading is catalyzed by Replication factor C (RFC) and the Ctf18 RFC-like complex (Ctf18-RLC), and its unloading is catalyzed by Atad5/Elg1-RLC. However, RFC, Ctf18-RLC, and even some subcomplexes of their shared subunits are capable of unloading PCNA in vitro, leaving an ambiguity in the division of labor in eukaryotic clamp dynamics. By using a system that specifically detects PCNA unloading, we show here that Atad5-RLC, which accounts for only approximately 3% of RFC/RLCs, nevertheless provides the major PCNA unloading activity in Xenopus egg extracts. RFC and Ctf18-RLC each account for approximately 40% of RFC/RLCs, while immunodepletion of neither Rfc1 nor Ctf18 detectably affects the rate of PCNA unloading in our system. PCNA unloading is dependent on the ATP-binding motif of Atad5, independent of nicks on DNA and chromatin assembly, and inhibited effectively by PCNA-interacting peptides. These results support a model in which Atad5-RLC preferentially unloads DNA-bound PCNA molecules that are free from their interactors.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas , Proteínas de Unión al ADN , Antígeno Nuclear de Célula en Proliferación , Animales , ADN , Replicación del ADN , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Xenopus laevis/metabolismo , Oocitos , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismoRESUMEN
OBJECTIVE: Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer that originates from the squamous cells. The role of the replication factor C subunit 3 (RFC3) in HNSCC progression remains elusive. The aim of this study was to uncover RFC3 significance in HNSCC. METHODS: The Cancer Genome Atlas (TCGA-HNSCC) dataset was initially used to assess RFC3 expression and its association with HNSCC clinical features. Subsequently, quantitative reverse transcription PCR (RT-qPCR) confirmed RFC3 mRNA expression in oral squamous cell carcinoma (OSCC), a primary HNSCC type. Survival rates were evaluated using the Kaplan-Meier plot, while the Tumor Immune Estimation Resource (TIMER) database probed RFC3-immune cell interaction. Additionally, in silico tools were used to examine the RFC3 protein network and engagement in HNSCC pathways. RESULTS: RFC3 expression is significantly upregulated in HNSCC, including OSCC. Upregulated RFC3 expression was significantly correlated with the clinicopathological features of HNSCC, including tumor stage, grade, metastasis, and patient survival. RFC3 is also associated with immune cell infiltration. Functional analysis has highlighted its involvement in DNA replication, mismatch repair, and cell cycle pathways. Interestingly, RFC3 high expression is linked to well-known oncogenic signaling pathways, such as MYC/MYCN, HIPPO, and mTOR. CONCLUSIONS: In conclusion, RFC3 can be considered a novel prognostic biomarker for HNSCC, and further studies on its functional mechanisms may help to use RFC3 as a therapeutic target for HNSCC. CLINICAL RELEVANCE: The clinical relevance of this study lies in identifying RFC3 as a novel biomarker and prognostic indicator for HNSCC, offering insights that could impact future clinical approaches.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias de la Boca , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello , Carcinoma de Células Escamosas/patología , Pronóstico , Proteína de Replicación C/metabolismoRESUMEN
BACKGROUND: Replication factor C subunit 2 (RFC2) participates in the growth and metastasis of various malignancies. Our study investigated the roles of RFC2 in colorectal cancer (CRC). RESULTS: RFC2 expression was upregulated in CRC tissues and cells. High RFC2 expression was associated with poor prognosis. Knockdown RFC2 inhibited proliferation, induced apoptosis, and suppressed migration and invasion of CRC cells. CREB5 was a transcription factor of RFC2, and CREB5 knockdown suppressed RFC2 expression. Furthermore, RFC2 promoted aerobic glycolysis and MET/PI3K/AKT/mTOR pathway. CONCLUSION: RFC2 promoted the progression of CRC cells via activating aerobic glycolysis and the MET/PI3K/AKT/mTOR pathway.
Asunto(s)
Neoplasias Colorrectales , Transducción de Señal , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína de Replicación C/metabolismo , Proliferación Celular/genética , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias Colorrectales/patología , Glucólisis , Línea Celular TumoralRESUMEN
Rad24-RFC (replication factor C) loads the 9-1-1 checkpoint clamp onto the recessed 5' ends by binding a 5' DNA at an external surface site and threading the 3' single-stranded DNA (ssDNA) into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' end, thus presumably leaving 9-1-1 on duplex 3' ss/double-stranded DNA (dsDNA) after Rad24-RFC ejects from DNA. We captured five Rad24-RFC-9-1-1 loading intermediates using a 10-nt gap DNA. We also determined the structure of Rad24-RFC-9-1-1 using a 5-nt gap DNA. The structures reveal that Rad24-RFC is unable to melt DNA ends and that a Rad24 loop limits the dsDNA length in the chamber. These observations explain Rad24-RFC's preference for a preexisting gap of over 5-nt ssDNA and suggest a direct role of the 9-1-1 in gap repair with various TLS (trans-lesion synthesis) polymerases in addition to signaling the ATR kinase.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Daño del ADN , ADN/metabolismo , Replicación del ADN , Proteína de Replicación C/metabolismo , Biología , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
Replication factor C 5 (RFC5) is involved in a variety of biological functions of cancer. However, the expression pattern of RFC5 and the underlying mechanisms in colorectal cancer (CRC) remain elusive. Here, we show that RFC5 is significantly upregulated in CRC tissues and cells. Patients with CRC and increased RFC5 levels have an unfavorable prognosis. RFC5 can promote the proliferation, migration, and invasion of CRC cells and inhibit the apoptosis of CRC cells. Additionally, upstream of RFC5, we constructed the competing endogenous RNA network and confirmed that RFC5 in this network was inhibited by miR-3614-5p by directly targeting its 3'-untranslated regions. We verified that circ_0038985, which is positively correlated with RFC5, directly targeted miR-3614-5p. Overexpression of circ_0038985 promoted CRC cell migration and invasion, and these effects were partially reversed by the reintroduction of miR-3614-5p. Moreover, we found that RFC5 may promote the vascular endothelial growth factor A (VEGFa)/vascular endothelial growth factor receptor 2 (VEGFR2)/extracellular signal-regulated protein kinase (ERK) pathway. The knockdown of RFC5 reduced CRC tumorigenesis in vivo. Collectively, these data demonstrate that the circ_0038985/miR-3614-5p/RFC5 axis plays a critical role in the progression of CRC, and RFC5 may promote CRC progression by affecting the VEGFa/VEGFR2/ERK pathway.
Asunto(s)
Neoplasias Colorrectales , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , OncogenesRESUMEN
Eukaryotic cells harbor two DNA-binding clamps, proliferating cell nuclear antigen (PCNA), and another clamp commonly referred to as 9-1-1 clamp. In contrast to the essential role of PCNA in DNA replication as a sliding clamp for DNA polymerase (Pol) δ, no such role in DNA synthesis has been identified for the human 9-1-1 clamp or the orthologous yeast 17-3-1 clamp. The only role identified for either the 9-1-1 or 17-3-1 clamp is in the recruitment of signal transduction kinases, which affect the activation of cell cycle checkpoints in response to DNA damage. However, unlike the loading of PCNA by the replication factor C (RFC) clamp loader onto 3'-recessed DNA junctions for processive DNA synthesis by Polδ, the 17-3-1 clamp or the 9-1-1 clamp is loaded by their respective clamp loader Rad24-RFC or RAD17-RFC onto the 5'-recessed DNA junction of replication protein A-coated DNA for the recruitment of signal transduction kinases. Here, we identify a novel role of 17-3-1 clamp as a sliding clamp for DNA synthesis by Polε. We provide evidence that similar to the loading of PCNA by RFC, the 17-3-1 clamp is loaded by the Rad24-RFC clamp loader at the 3'-recessed DNA junction in an ATP-dependent manner. However, unlike PCNA, the 17-3-1 clamp does not enhance the processivity of DNA synthesis by Polε; instead, it greatly increases the catalytic efficiency of Polε for correct nucleotide incorporation. Furthermore, we show that the same PCNA-interacting peptide domain in the polymerase 2 catalytic subunit mediates Polε interaction with the 17-3-1 clamp and with PCNA.