RESUMEN
Initiation of mitotic DNA replication in eukaryotes requires conserved factors, including Cdc18/CDC6 and minichromosome maintenance (MCM) proteins. We show here that these proteins are not essential for meiotic DNA replication or subsequent meiotic divisions in fission yeast. In addition, vegetative replication checkpoint genes are not required for the arrest of meiotic divisions in response to pre-meiotic S-phase delays. Genes essential for other aspects of vegetative DNA replication, however, including polymerases and DNA ligase, are also required for pre-meiotic DNA synthesis. Our results indicate that the process of replication initiation and checkpoint control may be fundamentally different in mitotic and meiotic cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Proteínas de Unión al ADN , Proteínas Fúngicas/metabolismo , Meiosis/fisiología , Mitosis/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Transglutaminasas , Proteínas de Ciclo Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , ADN de Hongos/biosíntesis , Diploidia , Proteínas Fúngicas/genética , Componente 4 del Complejo de Mantenimiento de Minicromosoma , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Recombinación Genética , Fase S , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/fisiologíaRESUMEN
MCM proteins are a conserved family of eukaryotic replication factors implicated in the initiation of DNA replication and in the discrimination between replicated and unreplicated chromatin. However, most mcm mutants in yeast arrest the cell cycle after bulk DNA synthesis has occurred. We investigated the basis for this late S phase arrest by analyzing the effects of a temperature-sensitive mutation in fission yeast cdc19(+ )(mcm2(+)). cdc19-P1 cells show a dramatic loss of viability at the restrictive temperature, which is not typical of all S phase mutants. The cdc19-P1 cell cycle arrest requires an intact damage-response checkpoint and is accompanied by increased rates of chromosome loss and mitotic recombination. Chromosomes from cdc19-P1 cells migrate aberrantly in pulsed-field gels, typical of strains arrested with unresolved replication intermediates. The cdc19-P1 mutation reduces the level of the Cdc19 protein at all temperatures. We compared the effects of disruptions of cdc19(+ )(mcm2(+)), cdc21(+ )(mcm4(+)), nda4(+ )(mcm5(+)) and mis5(+ )(mcm6(+)); in all cases, the null mutants underwent delayed S phase but were unable to proceed through the cell cycle. Examination of protein levels suggests that this delayed S phase reflects limiting, but not absent, MCM proteins. Thus, reduced dosage of MCM proteins allows replication initiation, but is insufficient for completion of S phase and cell cycle progression.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Fase S/genética , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Alelos , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , División Celular/genética , Replicación del ADN/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiología , Dosificación de Gen , Componente 4 del Complejo de Mantenimiento de Minicromosoma , Componente 6 del Complejo de Mantenimiento de Minicromosoma , Mutación , Fenotipo , Fase S/fisiología , Schizosaccharomyces/metabolismo , TemperaturaRESUMEN
The cdc19+ gene encodes an essential member of the MCM family of replication proteins in Schizosaccharomyces pombe. We have examined the structure and function of the Cdc19p protein using molecular and genetic approaches. We find that overproduction of wild-type Cdc19p in wild-type cells has no effect, but cdc19-P1 mutant cells do not tolerate elevated levels of other MCM proteins or overexpression of mutant forms of Cdc19p. We have found genetic interactions between cdc19+ and genes encoding subunits of DNA polymerase delta and the replication initiator cdc18+. We have constructed a series of point mutations and sequence deletions throughout Cdc19p, which allow us to distinguish essential from nonessential regions of the protein. Not surprisingly, conserved residues in the MCM homology domain are required for protein function, but some residues outside the core homology domain are dispensable.