RESUMO
Colorectal cancer (CRC) is one of the most common malignant carcinoma worldwide. DNA polymerase epsilon 2, accessory subunit (POLE2) participates in DNA replication, repair, and cell cycle control, but its association with CRC development remains unclear. In the present study, the differentially expressed genes (DEGs) in CRC were screened from bioinformatics analysis based on GEO database. RT-qPCR was used to assess mRNA expression. CCK-8 and colony formation assays were applied for the evaluation of cell proliferation. Wound healing and transwell assays were used to detect cell migration and invasion. Protein levels were determined by Western blotting assay. We found that POLE2 was highly expressed in CRC tissues and cell lines. Inhibition of POLE2 suppressed the proliferation, migration and invasion of CRC cells. Mechanistically, Wnt/ß-catenin signaling pathway was inactivated by inhibition of POLE2. Activation of Wnt/ß-catenin pathway can reverse the function of POLE2 knockdown on CRC cells. In vivo studies demonstrated that POLE2 silencing could notably inhibit the growth of tumors, which was consistent with the results in vitro. In conclusion, we found POLE2 as a novel oncogene in CRC, providing a potential therapeutic or diagnostic target in CRC.
Assuntos
Proliferação de Células , Neoplasias Colorretais , Via de Sinalização Wnt , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Camundongos , Animais , Movimento Celular , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Inativação Gênica , DNA Polimerase II/genética , DNA Polimerase II/metabolismoRESUMO
An organism's genomic DNA must be accurately duplicated during each cell cycle. DNA synthesis is catalysed by DNA polymerase enzymes, which extend nucleotide polymers in a 5' to 3' direction. This inherent directionality necessitates that one strand is synthesised forwards (leading), while the other is synthesised backwards discontinuously (lagging) to couple synthesis to the unwinding of duplex DNA. Eukaryotic cells possess many diverse polymerases that coordinate to replicate DNA, with the three main replicative polymerases being Pol α, Pol δ and Pol ε. Studies conducted in yeasts and human cells utilising mutant polymerases that incorporate molecular signatures into nascent DNA implicate Pol ε in leading strand synthesis and Pol α and Pol δ in lagging strand replication. Recent structural insights have revealed how the spatial organization of these enzymes around the core helicase facilitates their strand-specific roles. However, various challenging situations during replication require flexibility in the usage of these enzymes, such as during replication initiation or encounters with replication-blocking adducts. This review summarises the roles of the replicative polymerases in bulk DNA replication and explores their flexible and dynamic deployment to complete genome replication. We also examine how polymerase usage patterns can inform our understanding of global replication dynamics by revealing replication fork directionality to identify regions of replication initiation and termination.
Assuntos
Replicação do DNA , Humanos , DNA/metabolismo , DNA/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Animais , DNA Polimerase II/metabolismo , Eucariotos/enzimologia , Eucariotos/genética , DNA Polimerase III/metabolismo , Células Eucarióticas/metabolismo , Células Eucarióticas/enzimologia , DNA Polimerase I/metabolismoRESUMO
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.
Assuntos
Replicação do DNA , Antígeno Nuclear de Célula em Proliferação , Proteína de Replicação C , Humanos , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , DNA Polimerase II/metabolismo , DNA Polimerase II/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas Nucleares , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica , Proteína de Replicação C/metabolismo , Proteína de Replicação C/química , Domínios ProteicosAssuntos
Neoplasias Colorretais , DNA Polimerase III , DNA Polimerase II , Inibidores de Checkpoint Imunológico , Proteínas de Ligação a Poli-ADP-Ribose , Humanos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Inibidores de Checkpoint Imunológico/uso terapêutico , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , MutaçãoRESUMO
The clinical success of PARP1/2 inhibitors (PARPi) prompts the expansion of their applicability beyond homologous recombination deficiency. Here, we demonstrate that the loss of the accessory subunits of DNA polymerase epsilon, POLE3 and POLE4, sensitizes cells to PARPi. We show that the sensitivity of POLE4 knockouts is not due to compromised response to DNA damage or homologous recombination deficiency. Instead, POLE4 loss affects replication speed leading to the accumulation of single-stranded DNA gaps behind replication forks upon PARPi treatment, due to impaired post-replicative repair. POLE4 knockouts elicit elevated replication stress signaling involving ATR and DNA-PK. We find POLE4 to act parallel to BRCA1 in inducing sensitivity to PARPi and counteracts acquired resistance associated with restoration of homologous recombination. Altogether, our findings establish POLE4 as a promising target to improve PARPi driven therapies and hamper acquired PARPi resistance.
Assuntos
Proteína BRCA1 , DNA Polimerase II , Replicação do DNA , Inibidores de Poli(ADP-Ribose) Polimerases , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Humanos , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , DNA Polimerase II/metabolismo , DNA Polimerase II/genética , Replicação do DNA/efeitos dos fármacos , Dano ao DNA , Linhagem Celular Tumoral , Recombinação Homóloga/genética , Recombinação Homóloga/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genéticaAssuntos
Ciclina B1 , DNA Polimerase II , Neoplasias do Endométrio , Imuno-Histoquímica , Proteínas de Ligação a Poli-ADP-Ribose , Proteína Supressora de Tumor p53 , Humanos , Feminino , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Ciclina B1/genética , Ciclina B1/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Mutação , Pessoa de Meia-Idade , Idoso , AdultoRESUMO
The advent of PARP inhibitors (PARPis) has profoundly changed the treatment landscape of BRCA1/BRCA2-mutated cancers. Despite this, the development of resistance to these compounds has become a major challenge. Hence, a detailed understanding of the mechanisms underlying PARPi sensitivity is crucially needed. Here, we show that loss of the POLE3-POLE4 subunits of DNA polymerase epsilon (Polε) strongly sensitizes cancer cells to PARPis in a Polε level-independent manner. Loss of POLE3-POLE4 is not associated with defective RAD51 foci formation, excluding a major defect in homologous recombination. On the contrary, treatment with PARPis triggers replicative gap accumulation in POLE3-POLE4 knockout (KO) cells in a PRIMPOL-dependent manner. In addition to this, the loss of POLE3-POLE4 further sensitizes BRCA1-silenced cells to PARPis. Importantly, the knockdown of 53BP1 does not rescue PARPi sensitivity in POLE3-POLE4 KO cells, bypassing a common PARPi resistance mechanism and outlining a potential strategy to sensitize cancer cells to PARPis.
Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Humanos , Replicação do DNA/efeitos dos fármacos , Linhagem Celular Tumoral , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , DNA Polimerase II/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Rad51 Recombinase/metabolismoRESUMO
Recycling of parental histones is an important step in epigenetic inheritance. During DNA replication, DNA polymerase epsilon subunit DPB3/DPB4 and DNA replication helicase subunit MCM2 are involved in the transfer of parental histones to the leading and lagging strands, respectively. Single Dpb3 deletion (dpb3Δ) or Mcm2 mutation (mcm2-3A), which each disrupts one parental histone transfer pathway, leads to the other's predominance. However, the biological impact of the two histone transfer pathways on chromatin structure and DNA repair remains elusive. In this study, we used budding yeast Saccharomyces cerevisiae to determine the genetic and epigenetic outcomes from disruption of parental histone H3-H4 tetramer transfer. We found that a dpb3Δ mcm2-3A double mutant did not exhibit the asymmetric parental histone patterns caused by a single dpb3Δ or mcm2-3A mutation, suggesting that the processes by which parental histones are transferred to the leading and lagging strands are independent. Surprisingly, the frequency of homologous recombination was significantly lower in dpb3Δ, mcm2-3A and dpb3Δ mcm2-3A mutants, likely due to the elevated levels of free histones detected in the mutant cells. Together, these findings indicate that proper transfer of parental histones during DNA replication is essential for maintaining chromatin structure and that lower homologous recombination activity due to parental histone transfer defects is detrimental to cells.
Assuntos
Replicação do DNA , Histonas , Recombinação Homóloga , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Histonas/metabolismo , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Recombinação Homóloga/genética , Replicação do DNA/genética , Mutação , Cromatina/metabolismo , Cromatina/genética , DNA Polimerase II/metabolismo , DNA Polimerase II/genética , Epigênese Genética , Reparo do DNARESUMO
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.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase II/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Replicação ViralRESUMO
Alterations in the exonuclease domain of DNA polymerase ε cause ultramutated cancers. These cancers accumulate AGA>ATA transversions; however, their genomic features beyond the trinucleotide motifs are obscure. We analyze the extended DNA context of ultramutation using whole-exome sequencing data from 524 endometrial and 395 colorectal tumors. We find that G>T transversions in POLE-mutant tumors predominantly affect sequences containing at least six consecutive purines, with a striking preference for certain positions within polypurine tracts. Using this signature, we develop a machine-learning classifier to identify tumors with hitherto unknown POLE drivers and validate two drivers, POLE-E978G and POLE-S461L, by functional assays in yeast. Unlike other pathogenic variants, the E978G substitution affects the polymerase domain of Pol ε. We further show that tumors with POLD1 drivers share the extended signature of POLE ultramutation. These findings expand the understanding of ultramutation mechanisms and highlight peculiar mutagenic properties of polypurine tracts in the human genome.
Assuntos
Neoplasias Colorretais , DNA Polimerase II , Humanos , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Mutação/genética , Mutagênese , Neoplasias Colorretais/patologia , DNA Polimerase III/genética , Sequenciamento do Exoma , Proteínas de Ligação a Poli-ADP-Ribose/genéticaRESUMO
Endometrial carcinoma (EC) is a prevalent gynecological tumor in women, and its treatment and prevention are significant global health concerns. The mutations in DNA polymerase ε (POLE) are recognized as key features of EC and may confer survival benefits in endometrial cancer patients undergoing anti-PD-1/PD-L1 therapy. However, the anti-tumor mechanism of POLE mutations remains largely elusive. This study demonstrates that the hot POLE P286R mutation impedes endometrial tumorigenesis by inducing DNA breakage and activating the cGAS-STING signaling pathway. The POLE mutations were found to inhibit the proliferation and stemness of primary human EC cells. Mechanistically, the POLE mutants enhance DNA damage and suppress its repair through the interaction with DNA repair proteins, leading to genomic instability and the upregulation of cytoplasmic DNA. Additionally, the POLE P286R mutant also increases cGAS level, promotes TBK1 phosphorylation, and stimulates inflammatory gene expression and anti-tumor immune response. Furthermore, the POLE P286R mutation inhibits tumor growth and facilitates the infiltration of cytotoxic T cells in human endometrial cancers. These findings uncover a novel mechanism of POLE mutations in antagonizing tumorigenesis and provide a promising direction for effective cancer therapy.
Assuntos
DNA Polimerase II , Neoplasias do Endométrio , Feminino , Humanos , Carcinogênese/genética , Transformação Celular Neoplásica , DNA , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Neoplasias do Endométrio/genética , Mutação/genética , Proteínas de Ligação a Poli-ADP-Ribose/genéticaRESUMO
Germline pathogenic variants in the exonuclease domain of the replicative DNA polymerase Pol ε encoded by the POLE gene, predispose essentially to colorectal and endometrial tumors by inducing an ultramutator phenotype. It is still unclear whether all the POLE alterations influence similar strength tumorigenesis, immune microenvironment, and treatment response. In this review, we summarize the current understanding of the mechanisms and consequences of POLE mutations in human malignancies; we highlight the heterogeneity of mutation rate and cancer aggressiveness among POLE variants, propose some mechanistic basis underlining such heterogeneity, and discuss novel considerations for the choice and efficacy of therapies of POLE tumors.
Assuntos
DNA Polimerase II , Neoplasias do Endométrio , Feminino , Humanos , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Replicação do DNA , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Mutação em Linhagem Germinativa , Mutação/genética , Microambiente Tumoral , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologiaRESUMO
Discutem-se aqui as formas de encaminhamento de pacientes ao Hospital Adauto Botelho, localizado em Cariacica, Espírito Santo. A pesquisa se deu por meio de prontuários médicos datados desde a inauguração em 1954 e de depoimentos de pessoas que trabalharam lá durante a segunda metade do século XX. Foram analisados 102 prontuários e entrevistadas quatro pessoas. A pesquisa dos prontuários mostra forte inserção da Chefatura de Polícia no processo de internação. As falas dos entrevistados reiteram esse ponto, mostrando também a longa duração das internações. São as histórias de vida dos internos que dão o tom deste trabalho. Conclui-se, a partir delas, que o Hospital Adauto Botelho, mais que uma instituição de tratamento, era um espaço de confinamento.
This paper discusses the procedures for referring patients to Adauto Botelho Hospital, in Cariacica, Espírito Santo state, Brazil. The research is based on the medical records since its inauguration in 1954 and statements by people who worked there in the second half of the twentieth century. One hundred and two records were analyzed and four people were interviewed. The records revealed the active involvement of the Chief of Police in hospitalizations. The interviews corroborate this, while also showing the long duration of the hospitalizations. The tone of the paper is set by the life stories of the people hospitalized there. The conclusion is that this hospital served not so much for treatment as for confinement.