RESUMO
The spindle assembly checkpoint (SAC) monitors defects in kinetochore-microtubule attachment or lack of tension at kinetochores and arrests cells at prometaphase. In fission yeast, the double mutant between pot1Δ and the helicase-dead point mutant of the RecQ helicase Rqh1 gene (rqh1-hd) accumulates Rad51-dependent recombination intermediates at telomeres and enters mitosis with those intermediates. Here, we found that SAC-dependent prometaphase arrest occurred more frequently in pot1Δ rqh1-hd double mutants than in rqh1-hd single mutants. SAC-dependent prometaphase arrest also occurred more frequently in rqh1-hd single mutants after cells were released from DNA replication block compared to the rqh1-hd single mutant in the absence of exogenous insult to the DNA. In both cases, Mad2 foci persisted longer than usual at kinetochores, suggesting a defect in kinetochore-microtubule attachment. In pot1Δ rqh1-hd double mutants and rqh1-hd single mutants released from DNA replication block, SAC-dependent prometaphase arrest was suppressed by the removal of the recombination or replication intermediates. Our results indicate that the accumulation of recombination or replication intermediates induces SAC-dependent prometaphase arrest, possibly by affecting kinetochore-microtubule attachment.
Assuntos
DNA Ribossômico/genética , Genes cdc/genética , Rad51 Recombinase/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Fuso Acromático/genética , Telômero/genética , Animais , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos/genética , Cromossomos Fúngicos/genética , Reparo do DNA/genética , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Mitose/genética , Mitose/fisiologia , Mutação/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Pot1 is a single-stranded telomere-binding protein that is conserved from fission yeast to mammals. Deletion of Schizosaccharomyces pombe pot1(+) causes immediate telomere loss. S. pombe Rqh1 is a homolog of the human RecQ helicase WRN, which plays essential roles in the maintenance of genomic stability. Here, we demonstrate that a pot1Δ rqh1-hd (helicase-dead) double mutant maintains telomeres that are dependent on Rad51-mediated homologous recombination. Interestingly, the pot1Δ rqh1-hd double mutant displays a "cut" (cell untimely torn) phenotype and is sensitive to the antimicrotubule drug thiabendazole (TBZ). Moreover, the chromosome ends of the double mutant do not enter the pulsed-field electrophoresis gel. These results suggest that the entangled chromosome ends in the pot1Δ rqh1-hd double mutant inhibit chromosome segregation, signifying that Pot1 and Rqh1 are required for efficient chromosome segregation. We also found that POT1 knockdown, WRN-deficient human cells are sensitive to the antimicrotubule drug vinblastine, implying that some of the functions of S. pombe Pot1 and Rqh1 may be conserved in their respective human counterparts POT1 and WRN.