Premature Silencing of the Spindle Assembly Checkpoint Is Prevented by the Bub1-H2A-Sgo1-PP2A Axis in Saccharomyces cerevisiae

Abstract

The spindle assembly checkpoint (SAC) monitors mistakes in kinetochore-microtubule interaction and its activation prevents anaphase entry. The SAC remains active until all chromosomes have achieved bipolar attachment which applies tension on kinetochores. Our previous data in budding yeast Saccharomyces cerevisiae show that Ipl1/Aurora B kinase and a centromereassociated protein, Sgo1, are required to prevent SAC silencing prior to tension generation, but we believe that this regulatory network is incomplete. Bub1 kinase is one of the SAC components, and Bub1-dependent H2A phosphorylation triggers centromere recruitment of Sgo1 by H2A in yeast and human cells. Although yeast cells lacking the kinase domain of Bub1 show competent SAC activation, we found that the mutant cells fail to maintain a prolonged checkpoint arrest in the presence of tensionless attachment. Mutation of the Bub1 phosphorylation site in H2A also results in premature SAC silencing in yeast cells. Previous data indicate that Sgo1 protein binds to PP2A Rts1 , and we found that rts1D mutants exhibited premature SAC silencing as well. We further revealed that sgo1 mutants with abolished binding to H2A or PP2A Rts1 displayed premature SAC silencing. Together, our results suggest that, in budding yeast S. cerevisiae, the Bub1-H2A-Sgo1-PP2A Rts1 axis prevents SAC silencing and helps prolonged checkpoint arrest prior to tension establishment at kinetochores.

The phosphorylation of a kinetochore protein Dam1 by Aurora B/Ipl1 kinase promotes chromosome bipolar attachment in yeast

“…It is likely that the M to G1 transition occurs between 80 and 100 min because of the decrease of the population of large-budded cells. Thus, we speculate that Dam1 dephosphorylation occurs during anaphase, and our previous observation that the protein level of anaphase inhibitor Pds1 decreases during this window supports this speculation 24 .

Figure 1 The phosphorylation of Dam1 is Ipl1-dependent and cell cycle regulated.

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The E2F activators control multiple mitotic regulators and maintain genomic integrity through Sgo1 and BubR1

“…However, we did not observe a significant percentage of micronuclei in cells with siBubR1 (8.0%) compared with siSgo1 (10.8%). Together, these data suggest that inactivation of BubR1 or Sgo1 similarly lead to inactivation of the SAC in asynchronously-growing cells [ 82 ], suggesting inactivation of Sgo1 leads to CA through other mechanisms.…”

“…Recent work has shown that BubR1 helps recruit Sgo1 into centromeres and is part of a network that regulates premature SAC silencing [ 82 ]. To investigate whether Sgo1 and BubR1 play a role in the activation of the SAC in cells overexpressing the three E2Fs, we performed a micronucleus assay with siRNAs targeting BubR1 and Sgo1 as a measure for SAC dysfunction (Figure 6H and 6I ).…”

Checkpoint Proteins Bub1 and Bub3 Delay Anaphase Onset in Response to Low Tension Independent of Microtubule-Kinetochore Detachment

“…Whether microtubule-generated tension silences the SAC is under debate. Evidence obtained in several organisms suggests that tension may serve to inactivate SAC signaling ( Jin and Wang, 2013 ; Jin et al, 2017 ; Maresca and Salmon, 2009 ; Uchida et al, 2009 ; Wan et al, 2009 ). The super-resolution light microscopy of kinetochore components in Drosophila and human HeLa cells indicates that increased stretch within the kinetochore is correlated with SAC inactivation ( Maresca and Salmon, 2009 ; Uchida et al, 2009 ; Wan et al, 2009 ).…”