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Accurate partition of duplicated genetic material to the daughter cells during mitosis relies on the maintenance of the physical linkage (cohesion) between sister chromatids until their bipolar attachment to the mitotic spindle. In response to a single straying chromatid within a cell, a surveillance mechanism called the spindle checkpoint blocks the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C), stabilizes securin (an APC/C substrate and an inhibitor of separase), and delays the activation of separase. This in turn prevents cleavage of cohesin by separase, preserves sister chromatid cohesion, and delays the onset of anaphase. The protein kinase, Bub1, is a key component of the spindle checkpoint. Bub1 has an upstream function in regulating the kinetochore localization of Mad2 and other downstream checkpoint components. In addition, recent biochemical studies have shown that Bub1 directly phosphorylates the APC/C activator, Cdc20, and inhibits APC/C. Finally, Bub1 has a noncheckpoint function at the kinetochores and preserves centromeric cohesion through the MEI-S332/shugoshin family of proteins. Therefore, Bub1 performs multiple tasks in mitosis that ensure the proper inheritance of chromosomes.

Sister chromatids in mammalian cells remain attached mostly at their centromeres at metaphase because of the loss of cohesion along chromosome arms in prophase. Here, we report that Bub1 retains centromeric cohesion in mitosis of human cells. Depletion of Bub1 or Shugoshin (Sgo1) in HeLa cells by RNA interference causes massive missegregation of sister chromatids that originates at centromeres. Surprisingly, loss of chromatid cohesion in Bub1 and Sgo1 RNA-interference cells does not appear to require the full activation of separase but, instead, triggers a mitotic arrest that depends on Mad2 and Aurora B. Bub1 maintains the steady-state levels and centromeric localization of Sgo1. Therefore, Bub1 protects centromeric cohesion through Shugoshin in mitosis.

The initiation of DNA synthesis is thought to occur at sites bound by a heteromeric origin recognition complex (ORC). Previously, we have shown that in Drosophila, the level of the large subunit, ORC1, is modulated during cell cycle progression and that changes in ORC1 concentration alter origin utilization during development. Here, we investigate the mechanisms underlying cell cycle-dependent degradation of ORC1. We show that signals in the non-conserved N-terminal domain of ORC1 mediate its degradation upon exit from mitosis and in G1 phase by the anaphase-promoting complex (APC) in vivo. Degradation appears to be the result of direct action of the APC, as the N-terminal domain is ubiquitylated by purified APC in vitro. This regulated proteolysis is potent, sufficient to generate a normal temporal distribution of protein even when transcription of ORC1 is driven by strong constitutive promoters. These observations suggest that in Drosophila, ORC1 regulates origin utilization much as does Cdc6 in budding yeast.

Summary: Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs through two mechanisms in vertebrate cells: (1) phosphorylation and removal of cohesin from chromosome arms by mitotic kinases, including Plk1, during prophase, and (2) cleavage of centromeric cohesin by separase at the metaphase-anaphase transition. Bub1 and the MEI-S332/Shugoshin (Sgo1) family of proteins protect centromeric cohesin from mitotic kinases during prophase. We show that human Sgo1 binds to protein phosphatase 2A (PP2A). PP2A localizes to centromeres in a Bub1-dependent manner. The Sgo1–PP2A interaction is required for centromeric localization of Sgo1 and proper chromosome segregation in human cells. Depletion of Plk1 by RNA interference (RNAi) restores centromeric localization of Sgo1 and prevents chromosome missegregation in cells depleted of PP2A_Aα. Our findings suggest that Bub1 targets PP2A to centromeres, which in turn maintains Sgo1 at centromeres by counteracting Plk1-mediated chromosome removal of Sgo1. [Copyright &y& Elsevier]

The spindle checkpoint delays chromosome segregation in response to misaligned sister chromatids during mitosis, thus ensuring the fidelity of chromosome inheritance. Through binding to Cdc20, the Mad2 spindle checkpoint protein inhibits the target of this checkpoint, the ubiquitin protein ligase APC/CCdc20. We now show that without cofactor binding or covalent modification Mad2 adopts two distinct folded conformations at equilibrium (termed N1-Mad2 and N2-Mad2). The structure of N2-Mad2 has been determined by NMR spectroscopy. N2-Mad2 is much more potent in APC/C inhibition. Overexpression of a Mad2 mutant that specifically sequesters N2-Mad2 partially blocks checkpoint signaling in living cells. The two Mad2 conformers interconvert slowly in vitro, but interconversion is accelerated by a fragment of Mad1, an upstream regulator of Mad2. Our results suggest that the unusual two-state behavior of Mad2 is critical for spindle checkpoint signaling. [ABSTRACT FROM AUTHOR]