Sister chromatid cohesion depends primarily on cohesin linkages (19
). In budding yeast, an additional mechanism operates specifically at the rDNA locus of chromosome XII. Removal of both cohesive mechanisms requires APCCdc20
-dependent degradation of securin and the liberation of separase, which cleaves a subunit of cohesin and also promotes dissolution of rDNA cohesion by activating the phosphatase Cdc14 (5
). Although it is well established that the only critical role of APCCdc20
in cohesin removal is the promotion of securin degradation, it was not known if the destruction of additional targets is necessary for the resolution of rDNA. Our results demonstrate that efficient rDNA resolution also depends on APCCdc20
-dependent destruction of Clb5 and Dbf4.
Although delayed, rDNA segregation does eventually occur in cells expressing stabilized forms of Clb5 and Dbf4, indicating that the destruction of these proteins is not essential for rDNA segregation in mitosis. In meiosis, however, rDNA segregation fails completely in the presence of stabilized Clb5 and Dbf4, revealing a critical function for the inactivation of these kinases in meiotic anaphase.
Previous evidence that the phosphatase Cdc14 promotes rDNA segregation (5
) suggested that rDNA cohesion depends on the phosphorylation of Cdk1 substrates. Our results are consistent with this possibility and also suggest that rDNA resolution depends specifically on the dephosphorylation of Clb5-Cdk1 targets, as rDNA segregation was not significantly affected by Clb2 overexpression. Given the known timing of Clb5 and Clb2 destruction, this cyclin specificity supports a model (Fig. ) in which Clb5 destruction and Cdc14 activation trigger the dephosphorylation in early anaphase of specific Cdk1 targets involved in rDNA segregation. The significant levels of Clb2 that remain in the anaphase cell are apparently insufficient to maintain the phosphorylation of these targets in the face of separase-dependent Cdc14 activation. However, we speculate that Clb2-Cdk1 activity in anaphase does maintain the phosphorylation state of other Cdk1 targets involved in spindle disassembly and mitotic exit; the dephosphorylation of these targets occurs after anaphase as a result of Clb2 destruction, perhaps coupled with the increased Cdc14 activity that results from activation of the mitotic-exit network (30
). Stabilization of Clb2 therefore blocks mitotic exit but not rDNA segregation or other anaphase processes.
FIG. 7. Model of regulatory mechanisms governing chromosome segregation in budding yeast. Bulk chromosome separation occurs when cohesin is cleaved by separase, the activation of which results from the APCCdc20-dependent destruction of securin. Efficient removal (more ...)
What are the key targets of Clb5-Cdk1 in rDNA cohesion? The molecular basis of rDNA cohesion is not well understood but seems to depend on complex mechanisms involving topoisomerase II, sumoylation, condensin, and RNA polymerase I (5
). Clb5-Cdk1 may act through one or multiple substrates to govern these mechanisms. We identified several Clb5-specific Cdk1 substrates in our previous work (16
), but none of these appear to be a clear candidate for the control of rDNA cohesion. A significant future challenge will be the systematic identification of all Clb5-specific Cdk1 substrates, which should allow discovery of the critical Clb5 substrates involved in rDNA resolution and other anaphase processes.
We also identified the Cdc7-binding partner Dbf4 as a potential regulator of rDNA cohesion in budding yeast. Dbf4-Cdc7 is known primarily as a regulator of the initiation of DNA synthesis, and no mitotic roles have been described for this kinase. How might Dbf4-Cdc7 regulate rDNA cohesion? The fact that the DBF4
mutant has no apparent cell cycle defect alone but enhances the segregation delay from the CLB5
mutant suggests that one function of Dbf4-Cdc7 might be to enhance Clb5-Cdk1 function, either by increasing its intrinsic kinase activity or by enhancing its activity toward some Clb5-Cdk1 target. Alternatively, Dbf4-Cdc7 could promote cohesion by directly phosphorylating some component of the rDNA machinery. Notably, recent work suggests that Dbf4-Cdc7 helps promote DNA recombination in meiotic prophase and monopolar chromosome attachment in meiosis I (23
). Dbf4-Cdc7 might therefore be involved in the control of multiple processes other than DNA replication.
In fission yeast and higher eukaryotes, it is not clear if there are cohesin-independent chromosome linkages like those operating at budding-yeast rDNA. The fission yeast ortholog of Cdc14, Clp1, is not required for rDNA segregation (3
), but it remains possible that other phosphatases contribute to the dephosphorylation of Cdk1 substrates involved in rDNA cohesion. In mammalian cells, noncohesin rDNA linkages, if they exist, may be lost early in mitosis as a result of the “prophase” pathway that drives chromosome arm resolution and separation before metaphase (8
). Alternatively, rDNA resolution in animal cells might depend on the degradation of cyclin A in prometaphase. In some cell types, expression of APC-resistant cyclin A leads to a metaphase-like arrest with unseparated sister chromatids (6
), and cohesin-independent linkages may contribute to this separation defect.
Future models of mitosis should take into account the qualitative contributions of individual cyclin-Cdk1 complexes, in terms of both phosphorylation events early in the cell cycle and dephosphorylation events as cyclins are sequentially destroyed in late mitosis. Early destruction of cyclins that display strong substrate specificity, such as Clb5 and cyclin A, may be required for the correct timing of anaphase events. The substrate specificity of phosphatases such as Cdc14 provides another layer of complexity in the control of Cdk substrate dephosphorylation. These mechanisms collaborate to ensure that key Cdk substrates involved in processes such as chromosome segregation and spindle elongation are dephosphorylated at anaphase onset, while dephosphorylation of other Cdk substrates is delayed until later in mitosis.