RESUMEN
Cells experiencing DNA replication stress activate a response pathway that delays entry into mitosis and promotes DNA repair and completion of DNA replication. The protein kinases ScRad53 and SpCds1 (in baker's and fission yeast, respectively) are central to this pathway. We describe a conserved protein Mrc1, mediator of the replication checkpoint, required for activation of ScRad53 and SpCds1 during replication stress. mrc1 mutants are sensitive to hydroxyurea and have a checkpoint defect similar to rad53 and cds1 mutants. Mrc1 may be the replicative counterpart of Rad9 and Crb2, which are required for activating ScRad53 and Chk1 in response to DNA damage.
Asunto(s)
Replicación del ADN , ADN de Hongos/biosíntesis , Proteínas Fúngicas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2 , Activación Enzimática , Proteínas Fúngicas/genética , Genes Fúngicos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Proteínas Quinasas/metabolismo , Fase S , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombeRESUMEN
Yeast defective in the checkpoint kinase Rad53 fail to recover from transient DNA replication blocks and synthesize intact chromosomes. The effectors of Rad53 relevant to this recovery process are unknown. Here we report that overproduction of the chromatin assembly factor Asf1 can suppress the Ts phenotype of mrc1rad53 double mutants and the HU sensitivity of rad53 mutants. Eliminating silencing also suppresses this lethality, further implicating chromatin structure in checkpoint function. We find that Asf1 and Rad53 exist in a dynamic complex that dissociates in response to replication blocks and DNA damage. Thus, checkpoint pathways directly regulate chromatin assembly to promote survival in response to DNA damage and replication blocks.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2 , Daño del ADN , Replicación del ADN , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Genes Letales , Genes Supresores , Péptidos y Proteínas de Señalización Intracelular , Chaperonas Moleculares , Mutación , Nucleosomas/metabolismo , Proteínas Quinasas/genética , Temperatura , Levaduras/citología , Levaduras/efectos de los fármacos , Levaduras/genéticaRESUMEN
RAD53 and MEC1 are essential genes required for the transcriptional and cell cycle responses to DNA damage and DNA replication blocks. We have examined the essential function of these genes and found that their lethality but not their checkpoint defects can be suppressed by increased expression of genes encoding ribonucleotide reductase. Analysis of viable null alleles revealed that Mec1 plays a greater role in response to inhibition of DNA synthesis than Rad53. The loss of survival in mec1 and rad53 null or point mutants in response to transient inhibition of DNA synthesis is not a result of inappropriate anaphase entry but primarily to an inability to complete chromosome replication. We propose that this checkpoint pathway plays an important role in the maintenance of DNA synthetic capabilities when DNA replication is stressed.