Accurate chromosome segregation depends on sister kinetochores making bioriented attachments to microtubules from opposite poles. An essential regulator of biorientation is the Ipl1/Aurora B protein kinase that destabilizes improper microtubule-kinetochore attachments. To identify additional biorientation pathways, we performed a systematic genetic analysis between the ipl1-321 allele and all nonessential budding yeast genes. One of the mutants, mcm21Delta, precociously separates pericentromeres and this is associated with a defect in the binding of the Scc2 cohesin-loading factor at the centromere. Strikingly, Mcm21 becomes essential for biorientation when Ipl1 function is reduced, and this appears to be related to its role in pericentromeric cohesion. When pericentromeres are artificially tethered, Mcm21 is no longer needed for biorientation despite decreased Ipl1 activity. Taken together, these data reveal a specific role for pericentromeric linkage in ensuring kinetochore biorientation.

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The faithful segregation, or “partition”, of many low-copy-number bacterial plasmids is driven by plasmid-encoded ATPases that are represented by the P1 plasmid ParA protein. ParA binds to the bacterial nucleoid via an ATP-dependent non-specific DNA (nsDNA)1 binding activity, which is essential for partition. ParA also has a site-specific DNA binding activity to the par operator (parOP), which requires either ATP or ADP, and which is essential for it to act as a transcriptional repressor but is dispensable for partition. Here we examine how DNA binding by ParA contributes to the relative distribution of its plasmid partition and repressor activities, using a ParA with an alanine substitution at Arg351, a residue previously predicted to participate in site-specific DNA binding. In vivo, the parAR351A allele is compromised for partition, but its repressor activity is dramatically improved so that it behaves as a “super-repressor”. In vitro, ParAR351A binds and hydrolyzes ATP, and undergoes a specific conformational change required for nsDNA binding, but its nsDNA binding activity is significantly damaged. This defect in turn significantly reduces the assembly and stability of partition complexes formed by the interaction of ParA with ParB, the centromere-binding protein, and DNA. In contrast, the R351A change shows only a mild defect in site-specific DNA binding. We conclude that the partition defect is due to altered nsDNA binding kinetics and affinity for the bacterial chromosome. Further, the super-repressor phenotype is explained by an increased pool of non-nucleoid bound ParA that is competent to bind parOP and repress transcription.

The completion of chromosome segregation during anaphase requires the hypercondensation of the ϳ1-Mb rDNA array, a reaction dependent on condensin and Cdc14 phosphatase. Using systematic genetic screens, we identified 29 novel genetic interactions with budding yeast condensin. Of these, FOB1, CSM1, LRS4, and TOF2 were required for the mitotic condensation of the tandem rDNA array localized on chromosome XII. Interestingly, whereas Fob1 and the monopolin subunits Csm1 and Lrs4 function in rDNA condensation throughout M phase, Tof2 was only required during anaphase. We show that Tof2, which shares homology with the Cdc14 inhibitor Net1/Cfi1, interacts with Cdc14 phosphatase and its deletion suppresses defects in mitotic exit network (MEN) components. Consistent with these genetic data, the onset of Cdc14 release from the nucleolus was similar in TOF2 and tof2⌬ cells; however, the magnitude of the release was dramatically increased in the absence of Tof2, even when the MEN pathway was compromised. These data support a model whereby Tof2 coordinates the biphasic release of Cdc14 during anaphase by restraining a population of Cdc14 in the nucleolus after activation of the Cdc14 early anaphase release (FEAR) network, for subsequent release by the MEN. INTRODUCTIONFaithful chromosome transmission requires the precise coordination between chromosome segregation and cell division. In Saccharomyces cerevisiae, mitotic exit is tightly coupled with the late segregation of the repetitive ribosomal DNA (rDNA) locus through the timely release of the Cdc14 phosphatase, a key regulator of mitotic exit (for recent reviews, see D' Amours and Amon, 2004;Pereira and Schiebel, 2004). Cdc14 performs diverse functions at the closing of mitosis . First, Cdc14 promotes the late segregation of the ϳ1 Mb rDNA array through the anaphase recruitment of condensin, which serves to both resolve cohesin-independent linkages and compact the array Sullivan et al., 2004;Wang et al., 2004;Machin et al., 2005). Second, Cdc14 is required for anaphase spindle integrity through the localization of Sli15/Ipl1 kinase (Pereira and Schiebel, 2003). Finally, Cdc14 phosphatase functions to reset the cell cycle to the G1 state through the inactivation of mitotic cyclindependent kinases (CDK) and the reversal of CDK phosphorylation events (Visintin et al., 1998).Given these diverse functions, the spatiotemporal regulation of Cdc14 function is tightly controlled throughout the cell cycle. From G1 through anaphase onset, Cdc14 is sequestered in the nucleolus in a complex with its inhibitor Net1/Cfi1 Straight et al., 1999;Visintin et al., 1999;Traverso et al., 2001;Huang and Moazed, 2003). In budding yeast, the nucleolus is assembled around the ϳ1-1.5 Mb RDN locus, a highly specialized region owing to its repetitive nature, large size, and compartmentalization within the cell. The RDN locus comprises 100 -150 copies of the 9.1-kb ribosomal DNA repeat on chromosome XII (Petes, 1979). Each unit encodes the 35S and 5S ribosomal RNAs and contains two nontranscribed sp...