RESUMO
Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Cromátides/metabolismo , Cinetocoros/metabolismo , Animais , Antibacterianos/farmacologia , Proteínas de Ciclo Celular/genética , Linhagem Celular , Núcleo Celular/metabolismo , Galinhas , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA , Doxiciclina/farmacologia , Citometria de Fluxo , Proteínas Fúngicas , Humanos , Hibridização in Situ Fluorescente , Substâncias Macromoleculares , Microscopia de Força Atômica , Microscopia de Fluorescência , Proteínas Nucleares/metabolismo , Fenótipo , Fosfoproteínas , Subunidades Proteicas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae , CoesinasRESUMO
Cell division depends on the separation of sister chromatids in anaphase. In yeast, sister separation is initiated by cleavage of cohesin by the protease separase. In vertebrates, most cohesin is removed from chromosome arms by a cleavage-independent mechanism. Only residual amounts of cohesin are cleaved at the onset of anaphase, coinciding with its disappearance from centromeres. We have identified two separase cleavage sites in the human cohesin subunit SCC1 and have conditionally expressed noncleavable SCC1 mutants in human cells. Our results indicate that cohesin cleavage by separase is essential for sister chromatid separation and for the completion of cytokinesis.
Assuntos
Anáfase , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Cromossomos/metabolismo , Endopeptidases/metabolismo , Aneuploidia , Aurora Quinases , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Núcleo Celular/ultraestrutura , Centrômero/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona , Ciclina B/metabolismo , Replicação do DNA , Células HeLa , Humanos , Cariotipagem , Microscopia de Fluorescência , Microscopia de Vídeo , Mutação , Proteínas Nucleares , Fosfoproteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Separase , TransfecçãoRESUMO
Abnormalities of chromosome number are the most common genetic aberrations in cancer. The mechanisms regulating the fidelity of mitotic chromosome transmission in mammalian cells are therefore of great interest. Here we show that human cells without an hSecurin gene lose chromosomes at a high frequency. This loss was linked to abnormal anaphases during which cells underwent repetitive unsuccessful attempts to segregate their chromosomes. The abnormal mitoses were associated with biochemical defects in the activation of separin, the sister-separating protease, rendering it unable to cleave the cohesin subunit Scc1 efficiently. These results illuminate the function of mammalian securin and show that it is essential for the maintenance of euploidy.
Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Aberrações Cromossômicas/metabolismo , Cromossomos Humanos/metabolismo , Troca de Cromátide Irmã/fisiologia , Sequência de Aminoácidos , Anáfase/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrômero/genética , Centrômero/metabolismo , Aberrações Cromossômicas/genética , Transtornos Cromossômicos , Cromossomos Humanos/genética , Deleção de Genes , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese/fisiologia , Proteínas Nucleares , Fosfoproteínas , Proteínas de Saccharomyces cerevisiae , Fuso Acromático/metabolismoRESUMO
In yeast, anaphase depends on cohesin cleavage. How anaphase is controlled in vertebrates is unknown because their cohesins dissociate from chromosomes before anaphase. We show that residual amounts of the cohesin SCC1 remain associated with human centromeres until the onset of anaphase when a similarly small amount of SCC1 is cleaved. In Xenopus extracts, SCC1 cleavage depends on the anaphase-promoting complex and separin. Separin immunoprecipitates are sufficient to cleave SCC1, indicating that separin is associated with a protease activity. Separin activation coincides with securin destruction and partial separin cleavage, suggesting that several mechanisms regulate separin activity. We propose that in vertebrates, a cleavage-independent pathway removes cohesin from chromosome arms during prophase, whereas a separin-dependent pathway cleaves centromeric cohesin at the metaphase-anaphase transition.
