RESUMEN
Cohesin is a multiprotein complex essential for sister-chromatid cohesion. It plays a pivotal role in proper chromosome segregation and DNA damage repair. The mitotic behavior of cohesin is controlled through its phosphorylation, which possibly induces the dissociation of cohesin from chromosomes and enhances its susceptibility to separase. Here, we report using mass spectrometry and anti-phospho antibodies that the central domain of Rad21, the separase-target subunit of Schizosaccharomyces pombe cohesin, is regulated by various kinase-induced phosphorylation at nine residues, indicating the multiple roles for S. pombe cohesin. In vegetative and non-dividing G(0) cells, Rad21 is phosphorylated by unknown S/TP-consensus kinases, in mitotic and non-mitotic cells by polo/Plo1 and CDK, and in DNA-damaged cells by Rad3/ATR. While mitotic phosphorylation is implicated in the dissociation of Rad21 and its cleavage by separase in anaphase, the Rad3/ATR-dependent damage-induced phosphorylation occurs intensively at the time of repair completion, and only in post-replicative cells. This damage-induced Rad21 phosphorylation is involved in the recovery process of cells from checkpoint arrest, and needed for the removal of cohesin by separase after the completion of damage repair. These complex phospho-regulations of Rad21 indicate the functional significance of cohesin in cell adaptation to a variety of cellular conditions.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Reparación del ADN/fisiología , Endopeptidasas/metabolismo , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiología , Inmunoprecipitación de Cromatina , Inmunoquímica , Espectrometría de Masas , Fosforilación , Schizosaccharomyces/metabolismo , SeparasaRESUMEN
Centromeres contain specialized chromatin that includes the centromere-specific histone H3 variant, spCENP-A/Cnp1. Here we report identification of five fission yeast centromere proteins, Mis14-18. Mis14 is recruited to kinetochores independently of CENP-A, and, conversely, CENP-A does not require Mis14 to associate with centromeres. In contrast, Mis15, Mis16 (strong similarity with human RbAp48 and RbAp46), Mis17, and Mis18 are all part of the CENP-A recruitment pathway. Mis15 and Mis17 form an evolutionarily conserved complex that also includes Mis6. Mis16 and Mis18 form a complex and maintain the deacetylated state of histones specifically in the central core of centromeres. Mis16 and Mis18 are the most upstream factors in kinetochore assembly as they can associate with kinetochores in all kinetochore mutants except for mis18 and mis16, respectively. RNAi knockdown in human cells shows that Mis16 function is conserved as RbAp48 and RbAp46 are both required for localization of human CENP-A.
Asunto(s)
Proteínas Portadoras/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Acetilación , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Supervivencia Celular/genética , Centrómero/genética , Centrómero/ultraestructura , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Células HeLa , Histonas/genética , Humanos , Cinetocoros/metabolismo , Mutación/genética , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Interferencia de ARN , Proteína 4 de Unión a Retinoblastoma , Proteína 7 de Unión a Retinoblastoma , Schizosaccharomyces/genética , Schizosaccharomyces/ultraestructura , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/aislamiento & purificaciónRESUMEN
Sister chromatids are held together by cohesins. At anaphase, separase is activated by degradation of its inhibitory partner, securin. Separase then cleaves cohesins, thus allowing sister chromatid separation. Fission yeast securin (Cut2) has destruction boxes and a separase (Cut1) interaction site in the amino and carboxyl terminus, respectively. Here we show that securin is essential for separase stability and also for proper repair of DNA damaged by ultraviolet, X-ray and gamma-ray irradiation. The cut2(EA2) mutant is defective in the repair of ultraviolet damage lesions, although the DNA damage checkpoint is activated normally. In double mutant analysis of ultraviolet sensitivity, checkpoint kinase chk1 (ref. 9) and excision repair rad13 (ref. 10) mutants were additive with cut2(EA2), whereas recombination repair rhp51 (ref. 11) and cohesin subunit rad21 (ref. 12) mutants were not. Cohesin was hyper-modified on ultraviolet irradiation in a Rad3 kinase-dependent way. Experiments using either mutant cohesin that cannot be cleaved by separase or a protease-dead separase provide evidence that this DNA repair function of securin-separase acts through the cleavage of cohesin. We propose that the securin-separase complex might aid DNA repair by removing local cohesin in interphase cells.