S overexpressing CIK1-CC, we analyzed the cell cycle progression and chromosome segregation. To this finish, we introduced the PGAL CIK1-CC plasmid into WT and dam1?A cells with a GFP-marked centromere of chromosome IV (CEN4 FP) and mCherry-labeled microtubules (Tub1-mCherry). Following G 1 release into galactose medium, overexpression of CIK1-CC delayed cell cycle progression in WT cells as much more cells remained as large-budded presumably because of the induction of syntelic attachment, but this delay was not observed in dam1?A cells (Fig. 2A). We examined CEN4 FP segregation in WT and dam1?A mutant cells with an elongated spindle at 120 and 140 min. Overexpression of CIK1-CC only induced cosegregation of two CEN4 FP dots with a single spindle pole in three of WT cells, however the frequency of cosegregation improved to more than 30 in dam1?A mutant cells (Fig. 2A), probably causing viability loss. To further assess the checkpoint function in dam1?A cells in response to tension defects, we examined Pds1 protein levels, as its degradation marks anaphase onset. G1-arrested PDS1-18myc (c-Myc) and dam1-3A PDS1-18myc cells carrying either a vector or maybe a PGALCIK1-CC plasmid had been released into galactose medium. An obvious delay in Pds1 degradation was observed in WT cells overexpressing CIK1-CC, but this delay was abolished in dam1?A mutant cells (Fig.Formula of Methyl 2-amino-3-hydroxybenzoate 2B). Thus,dam1?A mutants are unable to delay anaphase entry in response to syntelic attachments. We also examined the checkpoint competency of dam1?A cells in response to tension defects induced by Mcd1 inactivation. Pds1 protein was stabilized in mcd1-1 cells incubated at 37 , but this stabilization was abolished in mad1, sgo1, and dam1?A mutant strains (Fig.916304-19-3 In stock S2A).PMID:25558565 Hence, tension defects induced by syntelic attachments or cohesin inactivation fail to delay anaphase onset in nonphosphorylatable dam1?A cells, supporting the conclusion that Dam1 phosphorylation by Ipl1 is required for the checkpoint response to tension defects. As ipl1 and sgo1 mutants show intact SAC function when treated with spindle poison nocodazole (7, 8), we compared the cell cycle progression in WT and dam1?A cells within the presence of nocodazole. Right after G1 release into the medium containing 20 g/mL of nocodazole, each WT and dam1?A cells arrested as large-budded cells with stabilized Pds1 (Fig. S2B), suggesting that the SAC is functional in dam1?A cells. Taken with each other, nonphosphorylatable dam1?A mutants behave like ipl1 and sgo1 mutants, which show intact SAC function but fail to arrest the cell cycle in response to tension defects.dam1?A Mutants Show Premature SAC Silencing in Response to Tension Defects. Mainly because our information indicate premature SAC si-lencing in ipl1 and sgo1 mutants in response to tension defects, we also analyzed the SAC silencing course of action in dam1?A cells. G1-arrested MAD1?HA and dam1?A MAD1-3HA cells carrying either a vector or PGALCIK1-CC plasmid had been released into galactose medium at 30 . The delayed Mad1 dephosphorylation induced by CIK1-CC overexpression was eliminated in dam1?A mutant cells (Fig. 3A). We also examined Mad1 phosphorylation kinetics in synchronized mcd1-1 and mcd1-1 dam1?A cells incubated at 37 . Clearly, dam1?A cells have been unable to maintain the phosphorylation status of Mad1 in the absence of tension (Fig. 3B), which may be a outcome of elevated Mad1 dephosphorylation or impaired Mad1 phosphorylation. Along with Mad1, other SAC proteins may well also turn into dephosphorylated ahead of SAC silencing. Ano.
