RESUMEN
Replication timing is a crucial aspect of genome regulation that is strongly correlated with chromatin structure, gene expression, DNA repair, and genome evolution. Replication timing is determined by the timing of replication origin firing, which involves activation of MCM helicase complexes loaded at replication origins. Nonetheless, how the timing of such origin firing is regulated remains mysterious. Here, we show that the number of MCMs loaded at origins regulates replication timing. We show for the first time in vivo that multiple MCMs are loaded at origins. Because early origins have more MCMs loaded, they are, on average, more likely to fire early in S phase. Our results provide a mechanistic explanation for the observed heterogeneity in origin firing and help to explain how defined replication timing profiles emerge from stochastic origin firing. These results establish a framework in which further mechanistic studies on replication timing, such as the strong effect of heterochromatin, can be pursued.
Asunto(s)
Momento de Replicación del ADN , Replicación del ADN , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Origen de Réplica , Ciclo Celular/genética , Inmunoprecipitación de Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
NO plays critical roles in vascular function. We show that modulation of the eNOS serine 1179 (S1179) phosphorylation site affects vascular reactivity and determines stroke size in vivo. Transgenic mice expressing only a phosphomimetic (S1179D) form of eNOS show greater vascular reactivity, develop less severe strokes, and have improved cerebral blood flow in a middle cerebral artery occlusion model than mice expressing an unphosphorylatable (S1179A) form. These results provide a molecular mechanism by which multiple diverse cardiovascular risks, such as diabetes and obesity, may be centrally integrated by eNOS phosphorylation in vivo to influence blood flow and cardiovascular disease. They also demonstrate the in vivo relevance of posttranslational modification of eNOS in vascular function.
Asunto(s)
Isquemia Encefálica/enzimología , Isquemia Encefálica/patología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Alanina/genética , Alanina/metabolismo , Animales , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Isquemia Encefálica/genética , Arterias Cerebrales/enzimología , Arterias Cerebrales/lesiones , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica , Masculino , Ratones , Ratones Transgénicos , Mutación/genética , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo III , Fosforilación , Flujo Sanguíneo Regional , Serina/genética , Serina/metabolismo , Accidente Cerebrovascular/enzimología , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/patologíaRESUMEN
Nitric oxide (NO) is a gaseous molecule that plays many key roles in the cardiovascular system. Each of the enzymes that generate NO--neuronal, inducible and endothelial NO synthase-has been genetically disrupted in mice. This review discusses the cardiovascular phenotypes of each of the NO synthase (NOS) gene knockout mice, and the insights gained into the roles of NO in the cardiovascular system. Mice lacking the endothelial isoform are hypertensive, have endothelial dysfunction and show a more severe outcome in response to vascular injury, to stroke and cerebral ischaemia, and to diet-induced atherosclerosis. Mice lacking the neuronal isoform show a less severe outcome in response to stroke and cerebral ischaemia but have increased diet-induced atherosclerosis. Mice lacking the inducible isoform show reduced hypotension to septic shock. Together, NOS gene knockout mice have been useful tools that complement our other approaches to studying the multiple roles of NO in the cardiovascular system.