RESUMEN
Stem cell division is linked to tumorigenesis by yet-elusive mechanisms. The hematopoietic system reacts to stress by triggering hematopoietic stem and progenitor cell (HSPC) proliferation, which can be accompanied by chromosomal breakage in activated hematopoietic stem cells (HSCs). However, whether these lesions persist in their downstream progeny and induce a canonical DNA damage response (DDR) remains unclear. Inducing HSPC proliferation by simulated viral infection, we report that the associated DNA damage is restricted to HSCs and that proliferating HSCs rewire their DDR upon endogenous and clastogen-induced damage. Combining transcriptomics, single-cell and single-molecule assays on murine bone marrow cells, we found accelerated fork progression in stimulated HSPCs, reflecting engagement of PrimPol-dependent repriming, at the expense of replication fork reversal. Ultimately, competitive bone marrow transplantation revealed the requirement of PrimPol for efficient HSC amplification and bone marrow reconstitution. Hence, fine-tuning replication fork plasticity is essential to support stem cell functionality upon proliferation stimuli.
Asunto(s)
Replicación del ADN , Hematopoyesis , Ratones , Animales , Hematopoyesis/genética , Células Madre Hematopoyéticas/fisiología , Daño del ADN , Proliferación CelularRESUMEN
Synthesis of deoxynucleoside triphosphates (dNTPs) is essential for both DNA replication and repair and a key step in this process is catalyzed by ribonucleotide reductases (RNRs), which reduce ribonucleotides (rNDPs) to their deoxy forms. Tight regulation of RNR is crucial for maintaining the correct levels of all four dNTPs, which is important for minimizing the mutation rate and avoiding genome instability. Although allosteric control of RNR was the first discovered mechanism involved in regulation of the enzyme, other controls have emerged in recent years. These include regulation of expression of RNR genes, proteolysis of RNR subunits, control of the cellular localization of the small RNR subunit, and regulation of RNR activity by small protein inhibitors. This review will focus on these additional mechanisms of control responsible for providing a balanced supply of dNTPs.
Asunto(s)
Reparación del ADN , Replicación del ADN , Ribonucleótido Reductasas/fisiología , Transporte Activo de Núcleo Celular , Animales , Ciclo Celular , Desoxirribonucleótidos/biosíntesis , Retroalimentación Fisiológica , Inestabilidad Genómica , HumanosRESUMEN
Aurora A kinase, a cell division regulator, is frequently overexpressed in various cancers, provoking genome instability and resistance to antimitotic chemotherapy. Localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. We have used fragment-based, structure-guided lead discovery to develop small molecule inhibitors of the Aurora A-TPX2 protein-protein interaction (PPI). Our lead compound, CAM2602, inhibits Aurora A:TPX2 interaction, binding Aurora A with 19 nM affinity. CAM2602 exhibits oral bioavailability, causes pharmacodynamic biomarker modulation, and arrests the growth of tumor xenografts. CAM2602 acts by a novel mechanism compared to ATP-competitive inhibitors and is highly specific to Aurora A over Aurora B. Consistent with our finding that Aurora A overexpression drives taxane resistance, these inhibitors synergize with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.
RESUMEN
The E3 ubiquitin ligase Cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) is emerging as an important cell cycle regulator that targets numerous proteins for destruction in S phase and after DNA damage, including Cdt1, p21, and Set8. CRL4(Cdt2) substrates contain a "PIP degron," which consists of a canonical proliferating cell nuclear antigen (PCNA) interaction motif (PIP box) and an adjacent basic amino acid. Substrates use their PIP box to form a binary complex with PCNA on chromatin and the basic residue to recruit CRL4(Cdt2) for substrate ubiquitylation. Using Xenopus egg extracts, we identify an acidic residue in PCNA that is essential to support destruction of all CRL4(Cdt2) substrates. This PCNA residue, which adjoins the basic amino acid of the bound PIP degron, is dispensable for substrate binding to PCNA but essential for CRL4(Cdt2) recruitment to chromatin. Our data show that the interaction of CRL4(Cdt2) with substrates requires molecular determinants not only in the substrate degron but also on PCNA. The results illustrate a potentially general mechanism by which E3 ligases can couple ubiquitylation to the formation of protein-protein interactions.
Asunto(s)
Antígeno Nuclear de Célula en Proliferación/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Xenopus/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Antígeno Nuclear de Célula en Proliferación/química , Unión Proteica , Schizosaccharomyces/metabolismo , Especificidad por Sustrato , Complejos de Ubiquitina-Proteína Ligasa , Ubiquitina-Proteína Ligasas/química , Proteínas de Xenopus/química , Xenopus laevis/metabolismoRESUMEN
Cdt1 plays a critical role in DNA replication regulation by controlling licensing. In Metazoa, Cdt1 is regulated by CRL4(Cdt2)-mediated ubiquitylation, which is triggered by DNA binding of proliferating cell nuclear antigen (PCNA). We show here that fission yeast Cdt1 interacts with PCNA in vivo and that DNA loading of PCNA is needed for Cdt1 proteolysis after DNA damage and in S phase. Activation of this pathway by ultraviolet (UV)-induced DNA damage requires upstream involvement of nucleotide excision repair or UVDE repair enzymes. Unexpectedly, two non-canonical PCNA-interacting peptide (PIP) motifs, which both have basic residues downstream, function redundantly in Cdt1 proteolysis. Finally, we show that poly-ubiquitylation of PCNA, which occurs after DNA damage, reduces Cdt1 proteolysis. This provides a mechanism for fine-tuning the activity of the CRL4(Cdt2) pathway towards Cdt1, allowing Cdt1 proteolysis to be more efficient in S phase than after DNA damage.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitinación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Cromatina/metabolismo , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/química , Datos de Secuencia Molecular , Fase S/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/efectos de la radiación , Proteínas de Schizosaccharomyces pombe/química , Rayos UltravioletaRESUMEN
Cells carrying the thermosensitive nrdA101 allele are able to replicate entire chromosomes at 42°C when new DNA initiation events are inhibited. We investigated the role of the recombination enzymes on the progression of the DNA replication forks in the nrdA101 mutant at 42°C in the presence of rifampin. Using pulsed-field gel electrophoresis (PFGE), we demonstrated that the replication forks stalled and reversed during the replication progression under this restrictive condition. DNA labeling and flow cytometry experiments supported this finding as the deleterious effects found in the RecB-deficient background were suppressed specifically by the absence of RuvABC; however, this did not occur in a RecG-deficient background. Furthermore, we show that the RecA protein is absolutely required for DNA replication in the nrdA101 mutant at restrictive temperature when the replication forks are reversed. The detrimental effect of the recA deletion is not related to the chromosomal degradation caused by the absence of RecA. The inhibition of DNA replication observed in the nrdA101 recA mutant at 42°C in the presence of rifampin was reverted by the presence of the wild-type RecA protein expressed ectopically but only partially suppressed by the RecA protein with an S25P mutation [RecA(S25P)], deficient in the rescue of the stalled replication forks. We propose that RecA is required to maintain the integrity of the reversed forks in the nrdA101 mutant under certain restrictive conditions, supporting the relationship between DNA replication and recombination enzymes through the stabilization and repair of the stalled replication forks.
Asunto(s)
Replicación del ADN , ADN Bacteriano/biosíntesis , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Rec A Recombinasas/metabolismo , Ribonucleósido Difosfato Reductasa/metabolismo , Antibacterianos/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Eliminación de Gen , Prueba de Complementación Genética , Calor , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Rec A Recombinasas/genética , Ribonucleósido Difosfato Reductasa/genética , Rifampin/metabolismoRESUMEN
NDP reductase activity can be inhibited either by treatment with hydroxyurea or by incubation of an nrdA (ts) mutant strain at the non-permissive temperature. Both methods inhibit replication, but experiments on these two types of inhibition yielded very different results. The chemical treatment immediately inhibited DNA synthesis but did not affect the cell and nucleoid appearance, while the incubation of an nrdA101 mutant strain at the non-permissive temperature inhibited DNA synthesis after more than 50 min, and resulted in aberrant chromosome segregation, long filaments, and a high frequency of anucleate cells. These phenotypes are not induced by SOS. In view of these results, we suggest there is an indirect relationship between NDP reductase and the chromosome segregation machinery through the maintenance of the proposed replication hyperstructure.
Asunto(s)
Segregación Cromosómica , Cromosomas Bacterianos/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/enzimología , Mutación/genética , Ribonucleósido Difosfato Reductasa/antagonistas & inhibidores , Temperatura , División Celular/efectos de los fármacos , División Celular/efectos de la radiación , Segregación Cromosómica/efectos de los fármacos , Segregación Cromosómica/efectos de la radiación , ADN Bacteriano/biosíntesis , Activación Enzimática/efectos de los fármacos , Activación Enzimática/efectos de la radiación , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Proteínas de Escherichia coli/metabolismo , Hidroxiurea/farmacología , Indoles , Luz , Ribonucleósido Difosfato Reductasa/metabolismo , Dispersión de RadiaciónRESUMEN
Analysis of accumulation of repair and checkpoint proteins at repair sites in yeast nuclei has conventionally used chemical agents, ionizing radiation or induction of endonucleases to inflict localized damage. In addition to these methods, similar studies in mammalian cells have used laser irradiation, which has the advantage that damage is inflicted at a specific nuclear region and at a precise time, and this allows accurate kinetic analysis of protein accumulation at DNA damage sites. We show here that it is feasible to use short pulses of near-infrared laser irradiation to inflict DNA damage in subnuclear regions of yeast nuclei by multiphoton absorption. In conjunction with use of fluorescently-tagged proteins, this allows quantitative analysis of protein accumulation at damage sites within seconds of damage induction. PCNA accumulated at damage sites rapidly, such that maximum accumulation was seen approximately 50 s after damage, then levels declined linearly over 200-1000 s after irradiation. RPA accumulated with slower kinetics such that hardly any accumulation was detected within 60 s of irradiation, and levels subsequently increased linearly over the next 900 s, after which levels were approximately constant (up to ca. 2700 s) at the damage site. This approach complements existing methodologies to allow analysis of key damage sensors and chromatin modification changes occurring within seconds of damage inception.
Asunto(s)
Daño del ADN/efectos de la radiación , Rayos Láser , Schizosaccharomyces/genética , Núcleo Celular/metabolismo , Reparación del ADN , Histonas/metabolismo , Rayos Infrarrojos , Microscopía Confocal , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína de Replicación A/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Imagen de Lapso de TiempoRESUMEN
Many individuals with multiple or large colorectal adenomas or early-onset colorectal cancer (CRC) have no detectable germline mutations in the known cancer predisposition genes. Using whole-genome sequencing, supplemented by linkage and association analysis, we identified specific heterozygous POLE or POLD1 germline variants in several multiple-adenoma and/or CRC cases but in no controls. The variants associated with susceptibility, POLE p.Leu424Val and POLD1 p.Ser478Asn, have high penetrance, and POLD1 mutation was also associated with endometrial cancer predisposition. The mutations map to equivalent sites in the proofreading (exonuclease) domain of DNA polymerases É and δ and are predicted to cause a defect in the correction of mispaired bases inserted during DNA replication. In agreement with this prediction, the tumors from mutation carriers were microsatellite stable but tended to acquire base substitution mutations, as confirmed by yeast functional assays. Further analysis of published data showed that the recently described group of hypermutant, microsatellite-stable CRCs is likely to be caused by somatic POLE mutations affecting the exonuclease domain.
Asunto(s)
Adenoma/genética , Neoplasias Colorrectales/genética , Reparación de la Incompatibilidad de ADN/genética , ADN Polimerasa III/genética , ADN Polimerasa II/genética , Replicación del ADN/genética , Modelos Moleculares , Exodesoxirribonucleasas/genética , Ligamiento Genético , Estudio de Asociación del Genoma Completo , Mutación de Línea Germinal/genética , Humanos , Repeticiones de Microsatélite/genética , Linaje , Proteínas de Unión a Poli-ADP-Ribosa , Schizosaccharomyces/genética , Análisis de Secuencia de ADNRESUMEN
Synthesis of deoxynucleoside triphosphates (dNTPs) is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimizing the mutation rate [3-7], and this is achieved by tight regulation of RNR [2, 8, 9]. In fission yeast, RNR is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow upregulation of dNTP supply [10-12]. Spd1 degradation is mediated by the activity of the CRL4(Cdt2) ubiquitin ligase complex [5, 13, 14]. This has been reported to be dependent on modulation of Cdt2 levels, which are cell cycle regulated, peaking in S phase, and which also increase after DNA damage in a checkpoint-dependent manner [7, 13]. We show here that Cdt2 level fluctuations are not sufficient to regulate Spd1 proteolysis and that the key step in this event is the interaction of Spd1 with the polymerase processivity factor proliferating cell nuclear antigen (PCNA), complexed onto DNA. This mechanism thus provides a direct link between DNA synthesis and RNR regulation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , ADN de Hongos/biosíntesis , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ribonucleótido Reductasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Datos de Secuencia Molecular , Mutación , Antígeno Nuclear de Célula en Proliferación/genética , Ribonucleótido Reductasas/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Brain-derived neurotrophic factor (BDNF) plays a critical role in the growth, differentiation and survival of neurons in the CNS. Recent research has suggested that BDNF may be implicated in the etiology of mood disorders and schizophrenia, as well as in the therapeutic action of some drugs, such as antidepressants and antipsychotics. This study aimed to develop a simple, fast and accurate new method for detecting the Val66Met polymorphism of the BDNF gene in schizophrenia patients using melting-curve analysis and a DNA-specific dye, SYBR Green I. A group of 30 schizophrenia patients were analyzed to detect the BDNF Val66Met polymorphism (rs6265) using the new genotyping method based on the analysis of fluorescence melting curves of PCR products that were labeled with SYBR Green I. The genotype results were confirmed for all 30 samples using the specific BDNF TaqMan allele discrimination assay. This new method allows the analysis of both alleles in the same reaction tube using SYBR Green I, with no need for additional steps. The addition of a GC clamp makes this method universally applicable, since the melting temperature of one allele can be adjusted as necessary to give the distinctive separation of melting curves. Therefore, this new method is simple, fast and accurate for determining the presence of the BDNF Val66Met polymorphism. It may also be useful for the analysis of other SNPs in pharmacogenetic studies.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Calor , Metionina/genética , Polimorfismo Genético , Valina/genética , Alelos , Benzotiazoles , ADN/genética , ADN/aislamiento & purificación , Diaminas , Colorantes Fluorescentes/metabolismo , Genotipo , Heterocigoto , Homocigoto , Humanos , Compuestos Orgánicos/metabolismo , Reacción en Cadena de la Polimerasa , Quinolinas , Esquizofrenia/etiología , Esquizofrenia/genética , Factores de Tiempo , Población Blanca/estadística & datos numéricosRESUMEN
The observed lengthening of the C period in the presence of a defective ribonucleoside diphosphate reductase has been assumed to be due solely to the low deoxyribonucleotide supply in the nrdA101 mutant strain. We show here that the nrdA101 mutation induces DNA double-strand breaks at the permissive temperature in a recB-deficient background, suggesting an increase in the number of stalled replication forks that could account for the slowing of replication fork progression observed in the nrdA101 strain in a Rec(+) context. These DNA double-strand breaks require the presence of the Holliday junction resolvase RuvABC, indicating that they have been generated from stalled replication forks that were processed by the specific reaction named "replication fork reversal." Viability results supported the occurrence of this process, as specific lethality was observed in the nrdA101 recB double mutant and was suppressed by the additional inactivation of ruvABC. None of these effects seem to be due to the limitation of the deoxyribonucleotide supply in the nrdA101 strain even at the permissive temperature, as we found the same level of DNA double-strand breaks in the nrdA(+) strain growing under limited (2-microg/ml) or under optimal (5-microg/ml) thymidine concentrations. We propose that the presence of an altered NDP reductase, as a component of the replication machinery, impairs the progression of the replication fork, contributing to the lengthening of the C period in the nrdA101 mutant at the permissive temperature.
Asunto(s)
Replicación del ADN , Escherichia coli/enzimología , Escherichia coli/fisiología , Ribonucleósido Difosfato Reductasa/genética , Ribonucleósido Difosfato Reductasa/fisiología , División Celular/genética , Recuento de Colonia Microbiana , Roturas del ADN de Doble Cadena , Replicación del ADN/genética , ADN Bacteriano/metabolismo , Desoxirribonucleótidos/biosíntesis , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Exodesoxirribonucleasa V/genética , Resolvasas de Unión Holliday/fisiología , Viabilidad Microbiana , MutaciónRESUMEN
Stalled replication forks produced by three different ways of depleting deoxynucleoside triphosphate showed different capacities to undergo "replication fork reversal." This reaction occurred at the stalled forks generated by hydroxyurea treatment, was impaired under thermal inactivation of ribonucleoside reductase, and did not take place under thymine starvation.