RESUMEN
Maintaining genomic integrity during DNA replication is essential for stem cells. DNA replication origins are licensed by the MCM2-7 complexes, with most of them remaining dormant. Dormant origins (DOs) rescue replication fork stalling in S phase and ensure genome integrity. However, it is not known whether DOs exist and play important roles in any stem cell type. Here, we show that embryonic stem cells (ESCs) contain more DOs than tissue stem/progenitor cells such as neural stem/progenitor cells (NSPCs). Partial depletion of DOs does not affect ESC self-renewal but impairs their differentiation, including toward the neural lineage. However, reduction of DOs in NSPCs impairs their self-renewal due to accumulation of DNA damage and apoptosis. Furthermore, mice with reduced DOs show abnormal neurogenesis and semi-embryonic lethality. Our results reveal that ESCs are equipped with more DOs to better protect against replicative stress than tissue-specific stem/progenitor cells.
Asunto(s)
Replicación del ADN , Células Madre Embrionarias/metabolismo , Inestabilidad Genómica , Células-Madre Neurales/metabolismo , Origen de Réplica , Estrés Fisiológico , Animales , Apoptosis , Línea Celular , Células Madre Embrionarias/citología , Ratones , Células-Madre Neurales/citología , NeurogénesisRESUMEN
Noncoding RNAs (ncRNAs) have crucial roles in epigenetic, transcriptional, and post-transcriptional regulation. Recent studies have begun to reveal a role of ncRNAs in DNA replication. Here, we review the roles of ncRNAs in regulating different aspects of DNA replication in prokaryotic and eukaryotic systems. We speculate that ncRNAs might function to guide the origin recognition complex (ORC) to chromosomal DNA during replication initiation in higher eukaryotes.
Asunto(s)
Replicación del ADN , Regulación de la Expresión Génica , ARN no Traducido/genética , Transcripción Genética , Animales , HumanosRESUMEN
Many replication origins that are licensed by loading MCM2-7 complexes in G1 are not normally used. Activation of these dormant origins during S phase provides a first line of defence for the genome if replication is inhibited. When replication forks fail, dormant origins are activated within regions of the genome currently engaged in replication. At the same time, DNA damage-response kinases activated by the stalled forks preferentially suppress the assembly of new replication factories, thereby ensuring that chromosomal regions experiencing replicative stress complete synthesis before new regions of the genome are replicated. Mice expressing reduced levels of MCM2-7 have fewer dormant origins, are cancer-prone and are genetically unstable, demonstrating the importance of dormant origins for preserving genome integrity. We review the function of dormant origins, the molecular mechanism of their regulation and their physiological implications.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Replicación del ADN/genética , Proteínas Nucleares/genética , Origen de Réplica/genética , Elementos Silenciadores Transcripcionales/genética , Animales , Proteínas de Unión al ADN/genética , Genes Supresores de Tumor , Ratones , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Componente 3 del Complejo de Mantenimiento de Minicromosoma , Componente 4 del Complejo de Mantenimiento de Minicromosoma , Componente 6 del Complejo de Mantenimiento de Minicromosoma , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Fase S/genética , Procesos EstocásticosRESUMEN
Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only â¼10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3-related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome.
Asunto(s)
Replicación del ADN/fisiología , Proteínas Quinasas/metabolismo , Origen de Réplica/fisiología , Afidicolina/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , Bromodesoxiuridina/farmacología , Cafeína/farmacología , Carbocianinas/metabolismo , Proteínas de Ciclo Celular/agonistas , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , ADN/metabolismo , Daño del ADN/fisiología , Replicación del ADN/efectos de los fármacos , Replicación del ADN/efectos de la radiación , Nucleótidos de Desoxiuracil/metabolismo , Fibroblastos/metabolismo , Rayos gamma , Humanos , Hidroxiurea/farmacología , Modelos Biológicos , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Purinas/farmacología , ARN Interferente Pequeño/genética , Origen de Réplica/efectos de los fármacos , Origen de Réplica/efectos de la radiación , RoscovitinaAsunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Replicación del ADN/fisiología , Animales , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , HumanosRESUMEN
Replication origins are 'licensed' for a single initiation event before entry into S phase; however, many licensed replication origins are not used, but instead remain dormant. The use of these dormant origins helps cells to survive replication stresses that block replication fork movement. Here, we present a computer model of the replication of a typical metazoan origin cluster in which origins are assigned a certain initiation probability per unit time and are then activated stochastically during S phase. The output of this model is in good agreement with experimental data and shows how inefficient dormant origins can be activated when replication forks are inhibited. The model also shows how dormant origins can allow replication to complete even if some forks stall irreversibly. This provides a simple explanation for how replication origin firing is regulated, which simultaneously provides protection against replicative stress while minimizing the cost of using large numbers of replication forks.
Asunto(s)
Simulación por Computador , Replicación del ADN/genética , Origen de Réplica/genética , Animales , HumanosRESUMEN
When DNA replication is slowed down, normally dormant replication origins are activated. Recent work demonstrates that cells adapt by changing the organization of chromatin loops and maintaining the new pattern of origin use in subsequent cell cycles.
Asunto(s)
Cromatina , Replicación del ADN , Origen de Réplica , Animales , Ciclo Celular , Células Eucariotas/citologíaRESUMEN
In late mitosis and early G1, Mcm2-7 complexes are loaded onto DNA to license replication origins for use in the upcoming S phase. However, the amount of Mcm2-7 loaded is in significant excess over the number of origins normally used. We show here that in human cells, excess chromatin-bound Mcm2-7 license dormant replication origins that do not fire during normal DNA replication, in part due to checkpoint activity. Dormant origins were activated within active replicon clusters if replication fork progression was inhibited, despite the activation of S-phase checkpoints. After lowering levels of chromatin-bound Mcm2-7 in human cells by RNA interference (RNAi), the use of dormant origins was suppressed in response to replicative stress. Although cells with lowered chromatin-bound Mcm2-7 replicated at normal rates, when challenged with replication inhibitors they had dramatically reduced rates of DNA synthesis and reduced viability. These results suggest that the use of dormant origins licensed by excess Mcm2-7 is a new and physiologically important mechanism that cells utilize to maintain DNA replication rates under conditions of replicative stress. We propose that checkpoint kinase activity can preferentially suppress initiation within inactive replicon clusters, thereby directing new initiation events toward active clusters that are experiencing replication problems.