RESUMEN
Minichromosome maintenance protein 10 (MCM10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 variants in patients with distinctive, but overlapping, clinical phenotypes: natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of MCM10-associated disease, we modeled these variants in human cell lines. MCM10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in MCM10-deficient cells require endonucleolytic processing by MUS81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic variants in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes observed in both NKD and RCM patients.
Asunto(s)
Alelos , Cardiomiopatías/genética , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/inmunología , Acortamiento del Telómero , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Humanos , Células Asesinas NaturalesRESUMEN
The ubiquitin-proteasome system is essential for cell cycle progression. Cyclin F is a cell cycle-regulated substrate adapter F-box protein for the Skp1, CUL1, and F-box protein (SCF) family of E3 ubiquitin ligases. Despite its importance in cell cycle progression, identifying cyclin F-bound SCF complex (SCFCyclin F) substrates has remained challenging. Since cyclin F overexpression rescues a yeast mutant in the cdc4 gene, we considered the possibility that other genes that genetically modify cdc4 mutant lethality could also encode cyclin F substrates. We identified the mitochondrial and cytosolic deacylating enzyme sirtuin 5 (SIRT5) as a novel cyclin F substrate. SIRT5 has been implicated in metabolic processes, but its connection to the cell cycle is not known. We show that cyclin F interacts with and controls the ubiquitination, abundance, and stability of SIRT5. We show SIRT5 knockout results in a diminished G1 population and a subsequent increase in both S and G2/M. Global proteomic analyses reveal cyclin-dependent kinase (CDK) signaling changes congruent with the cell cycle changes in SIRT5 knockout cells. Together, these data demonstrate that SIRT5 is regulated by cyclin F and suggest a connection between SIRT5, cell cycle regulation, and metabolism.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Proteínas F-Box/genética , Regulación Fúngica de la Expresión Génica , Procesamiento Proteico-Postraduccional , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sirtuinas/genética , Ubiquitina-Proteína Ligasas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Perfilación de la Expresión Génica , Genes Letales , Células HEK293 , Células HeLa , Humanos , Mutación , Proteínas Ligasas SKP Cullina F-box/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Sirtuinas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
To maintain tissue homeostasis, cells transition between cell cycle quiescence and proliferation. An essential G1 process is minichromosome maintenance complex (MCM) loading at DNA replication origins to prepare for S phase, known as origin licensing. A p53-dependent origin licensing checkpoint normally ensures sufficient MCM loading before S phase entry. We used quantitative flow cytometry and live cell imaging to compare MCM loading during the long first G1 upon cell cycle entry and the shorter G1 phases in the second and subsequent cycles. We discovered that despite the longer G1 phase, the first G1 after cell cycle re-entry is significantly underlicensed. Consequently, the first S phase cells are hypersensitive to replication stress. This underlicensing results from a combination of slow MCM loading with a severely compromised origin licensing checkpoint. The hypersensitivity to replication stress increases over repeated rounds of quiescence. Thus, underlicensing after cell cycle re-entry from quiescence distinguishes a higher-risk first cell cycle that likely promotes genome instability.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , División Celular/genética , Replicación del ADN/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Núcleo Celular/genética , Proliferación Celular/genética , Cromatina/genética , Citometría de Flujo , Fase G1/genética , Inestabilidad Genómica/genética , Humanos , Proteínas Nucleares/genética , Origen de Réplica/genética , Fase S/genéticaRESUMEN
Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.