RESUMEN
This case report elucidates a scenario involving two sibling sisters born out of consanguineous marriage-one initially presenting with lower respiratory infection, concurrently exhibiting short stature and primary amenorrhoea. Investigation into the primary amenorrhoea unveiled hypergonadotropic hypogonadism, confirmed by the absence of ovaries and a hypoplastic uterus on pelvic MRI. Genetic analysis via whole exome sequencing identified a homozygous variant NM_001282717.2: c.808C>T in the MCM8 gene, located on exon 8 of chromosome 20, inherited in an autosomal recessive manner. The scarcity of primary ovarian insufficiency cases linked to MCM8 highlights the necessity of thoroughly investigating the genetic and clinical consequences of such variants.
Asunto(s)
Proteínas de Mantenimiento de Minicromosoma , Mutación , Insuficiencia Ovárica Primaria , Hermanos , Útero , Humanos , Femenino , Insuficiencia Ovárica Primaria/genética , Proteínas de Mantenimiento de Minicromosoma/genética , Útero/anomalías , Consanguinidad , Imagen por Resonancia Magnética , Secuenciación del Exoma , Amenorrea/genética , Amenorrea/etiología , Anomalías Urogenitales/genética , Anomalías Urogenitales/diagnóstico por imagenRESUMEN
BACKGROUND: Gastric cancer (GC) is the fourth leading cause of cancer-related death worldwide. Minichromsome maintenance proteins family member 8 (MCM8) assists DNA repair and DNA replication. MCM8 exerts tumor promotor function in multiple digestive system tumors. MCM8 is also considered as a potential cancer therapeutic target. METHODS: Bioinformatics methods were used to analyze MCM8 expression and clinicopathological significance. MCM8 expression was detected by immunohistochemistry (IHC) staining and qRT-PCR. MCM8 functions in GC cell were explored by Celigo cell counting, colony formation, wound-healing, transwell, and annexin V-APC staining assays. The target of MCM8 was determined by human gene expression profile microarray. Human phospho-kinase array kit evaluated changes in key proteins after ribosomal protein S15A (RPS15A) knockdown. MCM8 functions were reassessed in xenograft mouse model. IHC detected related proteins expression in mouse tumor sections. RESULTS: MCM8 was significantly upregulated and predicted poor prognosis in GC. High expression of MCM8 was positively correlated with lymph node positive (p < 0.001), grade (p < 0.05), AJCC Stage (p < 0.001), pathologic T (p < 0.01), and pathologic N (p < 0.001). MCM8 knockdown inhibited proliferation, migration, and invasion while promoting apoptosis. RPS15A expression decreased significantly after MCM8 knockdown. It was also the only candidate target, which ranked among the top 10 downregulated differentially expressed genes (DEGs) in sh-MCM8 group. RPS15A was identified as the target of MCM8 in GC. MCM8/RPS15A promoted phosphorylation of P38α, LYN, and p70S6K. Moreover, MCM8 knockdown inhibited tumor growth, RPS15A expression, and phosphorylation of P38α, LYN, and p70S6K in vivo. CONCLUSIONS: MCM8 is an oncogene and predicts poor prognosis in GC. MCM8/RPS15A facilitates GC progression.
Asunto(s)
Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Proteínas Ribosómicas , Neoplasias Gástricas , Humanos , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidad , Animales , Ratones , Pronóstico , Femenino , Masculino , Línea Celular Tumoral , Progresión de la Enfermedad , Persona de Mediana Edad , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/genética , Apoptosis , Ratones Desnudos , Movimiento Celular , Ensayos Antitumor por Modelo de Xenoinjerto , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genéticaRESUMEN
MCM8 is a helicase, which participates in DNA replication and tumorigenesis and is upregulated in many human cancers, including lung cancer (LC); however, the function of MCM8 in LC tumour progression is unclear. In this study, we found that MCM8 was expressed at high levels in LC cells and tissues. Further, MCM8 upregulation was associated with advanced tumour grade and lymph node metastasis, and indicated poor prognosis. Silencing of MCM8 suppressed cell growth and migration in vitro and in vivo, while ectopic MCM8 expression promoted cell cycle progression, as well as cell migration, proliferation, and apoptosis. Mechanistically, DNAJC10 was identified as a downstream target of MCM8, using gene array and CO-IP assays. DNAJC10 overexpression combatted the inhibitory activity of MCM8 knockdown on LC progression, while silencing DNAJC10 alleviated the oncogenic function of MCM8 overexpression. MCM8 expression was positively correlated with that of DNAJC10 in LC samples from The Cancer Genome Atlas database, and DNAJC10 upregulation was also associated with poor overall survival of patients with LC. This study indicated that MCM8/DNAJC10 axis plays an important role in in LC development, and maybe as a new potential therapeutic target or a diagnostic biomarker for treating patients with LC.
Asunto(s)
Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares , Proteínas de Mantenimiento de Minicromosoma , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/genética , Proliferación Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Masculino , Animales , Femenino , Ratones , Apoptosis/genética , Regulación hacia Arriba/genética , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas del Choque Térmico HSP40/genética , Pronóstico , Ratones Desnudos , Persona de Mediana Edad , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genéticaRESUMEN
Eukaryotic DNA replication is a tightly controlled process that occurs in two main steps, i.e., licensing and firing, which take place in the G1 and S phases of the cell cycle, respectively. In Saccharomyces cerevisiae, the budding yeast, replication origins contain consensus sequences that are recognized and bound by the licensing factor Orc1-6, which then recruits the replicative Mcm2-7 helicase. By contrast, mammalian initiation sites lack such consensus sequences, and the mammalian ORC does not exhibit sequence specificity. Studies performed over the past decades have identified replication initiation sites in the mammalian genome using sequencing-based assays, raising the question of whether replication initiation occurs at confined sites or in broad zones across the genome. Although recent reports have shown that the licensed MCMs in mammalian cells are broadly distributed, suggesting that ORC-dependent licensing may not determine the initiation sites/zones, they are predominantly located upstream of actively transcribed genes. This review compares the mechanism of replication initiation in yeast and mammalian cells, summarizes the sequencing-based technologies used for the identification of initiation sites/zones, and proposes a possible mechanism of initiation-site/zone selection in mammalian cells. Future directions and challenges in this field are also discussed.
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Replicación del ADN , Origen de Réplica , Saccharomyces cerevisiae , Animales , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Complejo de Reconocimiento del Origen/genética , Mamíferos/genética , Genoma , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/genéticaRESUMEN
MCM8 has emerged as a core gene in reproductive aging and is crucial for meiotic homologous recombination repair. It also safeguards genome stability by coordinating the replication stress response during mitosis, but its function in mitotic germ cells remains elusive. Here we found that disabling MCM8 in mice resulted in proliferation defects of primordial germ cells (PGCs) and ultimately impaired fertility. We further demonstrated that MCM8 interacted with two known helicases DDX5 and DHX9, and loss of MCM8 led to R-loop accumulation by reducing the retention of these helicases at R-loops, thus inducing genome instability. Cells expressing premature ovarian insufficiency-causative mutants of MCM8 with decreased interaction with DDX5 displayed increased R-loop levels. These results show MCM8 interacts with R-loop-resolving factors to prevent R-loop-induced DNA damage, which may contribute to the maintenance of genome integrity of PGCs and reproductive reserve establishment. Our findings thus reveal an essential role for MCM8 in PGC development and improve our understanding of reproductive aging caused by genome instability in mitotic germ cells.
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ARN Helicasas DEAD-box , Inestabilidad Genómica , Proteínas de Mantenimiento de Minicromosoma , Estructuras R-Loop , Animales , Femenino , Humanos , Masculino , Ratones , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Daño del ADN , Células Germinativas/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/genética , Estructuras R-Loop/genéticaRESUMEN
To prevent detrimental chromosome re-replication, DNA loading of a double hexamer of the minichromosome maintenance (MCM) replicative helicase is temporally separated from DNA unwinding. Upon S-phase transition in yeast, DNA unwinding is achieved in two steps: limited opening of the double helix and topological separation of the two DNA strands. First, Cdc45, GINS and Polε engage MCM to assemble a double CMGE with two partially separated hexamers that nucleate DNA melting. In the second step, triggered by Mcm10, two CMGEs separate completely, eject the lagging-strand template and cross paths. To understand Mcm10 during helicase activation, we used biochemical reconstitution with cryogenic electron microscopy. We found that Mcm10 splits the double CMGE by engaging the N-terminal homo-dimerization face of MCM. To eject the lagging strand, DNA unwinding is started from the N-terminal side of MCM while the hexamer channel becomes too narrow to harbor duplex DNA.
Asunto(s)
Microscopía por Crioelectrón , Replicación del ADN , Proteínas de Mantenimiento de Minicromosoma , Origen de Réplica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructura , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/química , Modelos Moleculares , ADN de Hongos/metabolismo , ADN de Hongos/química , Multimerización de ProteínaRESUMEN
The helicase MCM and the ribonucleotide reductase RNR are the complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication. Here, we demonstrate that MCM interacts physically with RNR and some of its regulators, including the kinase Dun1. These physical interactions encompass small subpopulations of MCM and RNR, are independent of the major subcellular locations of these two complexes, augment in response to DNA damage and, in the case of the Rnr2 and Rnr4 subunits of RNR, depend on Dun1. Partial disruption of the MCM/RNR interactions impairs the release of Rad52 -but not RPA-from the DNA repair centers despite the lesions are repaired, a phenotype that is associated with hypermutagenesis but not with alterations in the levels of dNTPs. These results suggest that a specifically regulated pool of MCM and RNR complexes plays non-canonical roles in genetic stability preventing persistent Rad52 centers and hypermutagenesis.
Asunto(s)
Proteínas de Ciclo Celular , Daño del ADN , Reparación del ADN , Replicación del ADN , Inestabilidad Genómica , Proteína Recombinante y Reparadora de ADN Rad52 , Ribonucleótido Reductasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicación del ADN/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Daño del ADN/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/metabolismo , Reparación del ADN/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/genética , Proteína de Replicación A/metabolismo , Proteína de Replicación A/genética , Ribonucleósido Difosfato Reductasa/genética , Ribonucleósido Difosfato Reductasa/metabolismoRESUMEN
HROB promotes the MCM8-9 helicase in DNA damage response. To understand how HROB activates MCM8-9, we defined their interaction interface. We showed that HROB makes important yet transient contacts with both MCM8 and MCM9, and binds the MCM8-9 heterodimer with the highest affinity. MCM8-9-HROB prefer branched DNA structures, and display low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexamer that assembles from dimers on DNA in the presence of ATP. The hexamer involves two repeating protein-protein interfaces between the alternating MCM8 and MCM9 subunits. One of these interfaces is quite stable and forms an obligate heterodimer across which HROB binds. The other interface is labile and mediates hexamer assembly, independently of HROB. The ATPase site formed at the labile interface contributes disproportionally more to DNA unwinding than that at the stable interface. Here, we show that HROB promotes DNA unwinding downstream of MCM8-9 loading and ring formation on ssDNA.
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Reparación del ADN , Proteínas de Unión al ADN , Proteínas de Mantenimiento de Minicromosoma , Humanos , Adenosina Trifosfato/metabolismo , ADN/metabolismo , ADN/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/genética , Unión Proteica , Multimerización de Proteína , Reparación del ADN/genéticaRESUMEN
Germline-specific genes are usually activated in cancer cells and drive cancer progression; such genes are called cancer-germline or cancer-testis genes. The RNA-binding protein DAZL is predominantly expressed in germ cells and plays a role in gametogenesis as a translational activator or repressor. However, its expression and role in non-small cell lung cancer (NSCLC) are unknown. Here, mining of RNA-sequencing data from public resources and immunohistochemical analysis of tissue microarrays showed that DAZL was expressed exclusively in testis among normal human tissues but ectopically expressed in NSCLC tissues. Testis and NSCLC cells expressed the shorter and longer transcript variants of the DAZL gene, respectively. Overexpression of the longer DAZL transcript promoted tumor growth in a mouse xenograft model. Silencing of DAZL suppressed cell proliferation, colony formation, migration, invasion, and cisplatin resistance in vitro and tumor growth in vivo. Quantitative proteomic analysis based on tandem mass tag and Western blot analysis showed that DAZL upregulated the expression of JAK2 and MCM8. RNA-binding protein immunoprecipitation assays showed that DAZL bound to the mRNA of JAK2 and MCM8. The JAK2 inhibitor fedratinib attenuated the oncogenic outcomes induced by DAZL overexpression, whereas silencing MCM8 counteracted the effects of DAZL overexpression on cisplatin-damaged DNA synthesis and half-maximal inhibitory concentration of cisplatin. In conclusion, DAZL was identified as a novel cancer-germline gene that enhances the translation of JAK2 and MCM8 to promote NSCLC progression and resistance to cisplatin, respectively. These findings suggest that DAZL is a potential therapeutic target in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Cisplatino , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Janus Quinasa 2 , Neoplasias Pulmonares , Proteínas de Mantenimiento de Minicromosoma , Proteínas de Unión al ARN , Animales , Femenino , Humanos , Masculino , Ratones , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origins is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases, DNA polymerase α-primase, DNA polymerase δ and ε, at replication forks play pivotal roles in the mechanism of the initiation reactions of leading and lagging strand DNA synthesis. These interactions are also important for the initiation of signalling at unperturbed and stalled replication forks, "replication stress" events, via ATR (ATM-Rad 3-related protein kinase). These processes are essential for the accurate transfer of the cells' genetic information to their daughters. Thus, failures and dysfunctions in these processes give rise to genome instability causing genetic diseases, including cancer. In their influential review "Hallmarks of Cancer: New Dimensions", Hanahan and Weinberg (2022) therefore call genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels, which will be discussed in the review.
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Replicación del ADN , ADN , Humanos , ADN/genética , ADN/metabolismo , Replicación del ADN/genética , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Inestabilidad GenómicaRESUMEN
The minichromosome maintenance (MCM) protein is a component of an active helicase that is essential for the initiation of DNA replication. Dysregulation of MCM functions contribute to abnormal cell proliferation and genomic instability. The interactions of MCM with cellular factors, including Cdc45 and GINS, determine the formation of active helicase and functioning of helicase. The functioning of MCM determines the fate of DNA replication and, thus, genomic integrity. This complex is upregulated in precancerous cells and can act as an important tool for diagnostic applications. The MCM protein complex can be an important broad-spectrum therapeutic target in various cancers. Investigations have supported the potential and applications of MCM in cancer diagnosis and its therapeutics. In this article, we discuss the physiological roles of MCM and its associated factors in DNA replication and cancer pathogenesis.
Asunto(s)
Replicación del ADN , Proteínas de Mantenimiento de Minicromosoma , Neoplasias , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/genética , Inestabilidad Genómica , Biomarcadores de Tumor/metabolismo , ADN Helicasas/metabolismo , ADN Helicasas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , AnimalesRESUMEN
BACKGROUND: A high expression pattern of minichromosome maintenance 2 (MCM2) has been observed in various cancers. MCM2 is a protein involved in the cell cycle and plays a role in cancer growth and differentiation by binding to six members of the MCM subfamily. The MCM protein family includes MCM2 through MCM7. METHODS: MCM2 has shown high expression in both lung cancer stem cells (LCSCs) and glioma stem cells (GSCs). We investigated the characteristics of CSCs and the regulation of the epithelial-to-mesenchymal transition (EMT) phenomenon in LCSCs and GSCs by MCM2. Additionally, we explored secreted factors regulated by MCM2. RESULTS: There was a significant difference in survival rates between lung cancer patients and brain cancer patients based on MCM2 expression. MCM2 was found to regulate both markers and regulatory proteins in LCSCs. Moreover, MCM2 is thought to be involved in cancer metastasis by regulating cell migration and invasion, not limited to lung cancer but also identified in glioma. Among chemokines, chemokine (C-X-C motif) ligand 1 (CXCL1) was found to be regulated by MCM2. CONCLUSIONS: MCM2 not only participates in the cell cycle but also affects cancer cell growth by regulating the external microenvironment to create a favorable environment for cells. MCM2 is highly expressed in malignant carcinomas, including CSCs, and contributes to the malignancy of various cancers. Therefore, MCM2 may represent a crucial target for cancer therapeutics.
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Neoplasias Pulmonares , Proteínas de Mantenimiento de Minicromosoma , Humanos , Quimiocina CXCL1 , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas , Células Madre Neoplásicas/metabolismo , Componente 2 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Ciclo Celular/genética , Microambiente TumoralRESUMEN
Malignant tumors constitute a significant category of diseases posing a severe threat to human survival and health, thereby representing one of the most challenging and pressing issues in the field of biomedical research. Due to their malignant nature, which is characterized by a high potential for metastasis, rapid dissemination, and frequent recurrence, the prevailing approach in clinical oncology involves a comprehensive treatment strategy that combines surgery with radiotherapy, chemotherapy, targeted drug therapies, and other interventions. Treatment resistance remains a major obstacle in the comprehensive management of tumors, serving as a primary cause for the failure of integrated tumor therapies and a critical factor contributing to patient relapse and mortality. The Minichromosome Maintenance (MCM) protein family comprises functional proteins closely associated with the development of resistance in tumor therapy.The influence of MCMs manifests through various pathways, encompassing modulation of DNA replication, cell cycle regulation, and DNA damage repair mechanisms. Consequently, this leads to an enhanced tolerance of tumor cells to chemotherapy, targeted drugs, and radiation. Consequently, this review explores the specific roles of the MCM family in various cancer treatment strategies. Its objective is to enhance our comprehension of resistance mechanisms in tumor therapy, thereby presenting novel targets for clinical research aimed at overcoming resistance in cancer treatment. This bears substantial clinical relevance.
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Neoplasias , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Replicación del ADN , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMEN
Newly copied sister chromatids are tethered together by the cohesin complex, but how sister chromatid cohesion coordinates with DNA replication is poorly understood. Prevailing models suggest that cohesin complexes, bound to DNA before replication, remain behind the advancing replication fork to keep sister chromatids together. By visualizing single replication forks colliding with preloaded cohesin complexes, we find that the replisome instead pushes cohesin to where a converging replisome is met. Whereas the converging replisomes are removed during DNA replication termination, cohesin remains on nascent DNA and provides cohesion. Additionally, we show that CMG (CDC45-MCM2-7-GINS) helicase disassembly during replication termination is vital for proper cohesion in budding yeast. Together, our results support a model wherein sister chromatid cohesion is established during DNA replication termination.
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Cromátides , Cohesinas , Replicación del ADN , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Intercambio de Cromátides Hermanas , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cohesinas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1. Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, albeit with reduced affinity. Consistently, either surface is sufficient for replication origin firing in Xenopus egg extracts and becomes essential in the absence of the other. The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Polε) to GINS when bound to Mcm2-7-Cdc45, although TopBP1-BRCT4 and the Polε subunit PolE2 show only partial competitivity in binding to Psf1. Our TopBP1-GINS model improves the understanding of the recently characterised metazoan pre-loading complex. It further predicts the coordination of three molecular origin firing processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.
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Replicación del ADN , Proteínas de Unión al ADN , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa II/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Replicación ViralRESUMEN
Accurate and complete replication of genetic information is a fundamental process of every cell division. The replication licensing is the first essential step that lays the foundation for error-free genome duplication. During licensing, minichromosome maintenance protein complexes, the molecular motors of DNA replication, are loaded to genomic sites called replication origins. The correct quantity and functioning of licensed origins are necessary to prevent genome instability associated with severe diseases, including cancer. Here, we delve into recent discoveries that shed light on the novel functions of licensed origins, the pathways necessary for their proper maintenance, and their implications for cancer therapies.
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Replicación del ADN , Neoplasias , Humanos , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Neoplasias/genéticaRESUMEN
Natural killer (NK) cell deficiency (NKD) is a rare disease in which NK cell function is reduced, leaving affected individuals susceptible to repeated viral infections and cancer. Recently, a patient with NKD was identified carrying compound heterozygous variants of MCM10 (minichromosome maintenance protein 10), an essential gene required for DNA replication, that caused a significant decrease in the amount of functional MCM10. NKD in this patient presented as loss of functionally mature late-stage NK cells. To understand how MCM10 deficiency affects NK cell development, we generated MCM10 heterozygous (MCM10+/-) induced pluripotent stem cell (iPSC) lines. Analyses of these cell lines demonstrated that MCM10 was haploinsufficient, similar to results in other human cell lines. Reduced levels of MCM10 in mutant iPSCs was associated with impaired clonogenic survival and increased genomic instability, including micronuclei formation and telomere erosion. The severity of these phenotypes correlated with the extent of MCM10 depletion. Significantly, MCM10+/- iPSCs displayed defects in NK cell differentiation, exhibiting reduced yields of hematopoietic stem cells (HSCs). Although MCM10+/- HSCs were able to give rise to lymphoid progenitors, these did not generate mature NK cells. The lack of mature NK cells coincided with telomere erosion, suggesting that NKD caused by these MCM10 variants arose from the accumulation of genomic instability including degradation of chromosome ends.
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Células Madre Pluripotentes Inducidas , Humanos , Diferenciación Celular , Genes Esenciales , Inestabilidad Genómica , Células Asesinas Naturales , Proteínas de Mantenimiento de MinicromosomaRESUMEN
FANCJ, a DNA helicase linked to Fanconi anemia and frequently mutated in cancers, counteracts replication stress by dismantling unconventional DNA secondary structures (such as G-quadruplexes) that occur at the DNA replication fork in certain sequence contexts. However, how FANCJ is recruited to the replisome is unknown. Here, we report that FANCJ directly binds to AND-1 (the vertebrate ortholog of budding yeast Ctf4), a homo-trimeric protein adaptor that connects the CDC45/MCM2-7/GINS replicative DNA helicase with DNA polymerase α and several other factors at DNA replication forks. The interaction between FANCJ and AND-1 requires the integrity of an evolutionarily conserved Ctf4-interacting protein (CIP) box located between the FANCJ helicase motifs IV and V. Disruption of the CIP box significantly reduces FANCJ association with the replisome, causing enhanced DNA damage, decreased replication fork recovery and fork asymmetry in cells unchallenged or treated with Pyridostatin, a G-quadruplex-binder, or Mitomycin C, a DNA inter-strand cross-linking agent. Cancer-relevant FANCJ CIP box variants display reduced AND-1-binding and enhanced DNA damage, a finding that suggests their potential role in cancer predisposition.
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ADN , Neoplasias , Humanos , ADN/química , Replicación del ADN , Inestabilidad Genómica , Proteínas de Mantenimiento de MinicromosomaRESUMEN
BACKGROUND: Head and neck squamous cell carcinoma (HNSC) is a common malignant tumor in the world and has a poor prognosis. The family of minichromosome maintenance proteins (MCM) improves the stability of genome replication by inhibiting the rate of DNA replication in eukaryotic cells, thus, small changes in physiological MCM levels would increase the instability of gene replication and increase the incidence of tumor formation, most of which are significantly elevated in multiple cancers. However, the expression of different MCM families in HNSC and their prognostic value remain unclear. METHODS: ONCOMINE and GEPIA databases were used to analyze the expression of MCMs in HNSC. The Kaplan-Meier plotter database was used to identify molecules with prognostic values. We collected 77 HNSC tissues and 50 normal tissues to validate the results of the bioinformatics analysis by immunohistochemical staining. RESULTS: The expression of MCM3, MCM5 and MCM6 in mRNA and protein levels were higher in HNSC. Moreover, the increased expression of MCM3, MCM5 and MCM6 in mRNA and protein levels predicted better prognosis of HNSC patients. Furthermore, multivariate analysis showed that high expressions of MCM3, MCM5 and MCM6 in protein level may be independent prognostic factors for HNSC patients. CONCLUSION: The results of this study indicated that MCM3, MCM5 and MCM6 play an important role in occurrence and development in HNSC and might be risk factors for the survival of HNSC patients.