Polo Kinase Regulates Mitotic Chromosome Condensation by Hyperactivation of Condensin DNA Supercoiling Activity

Molecular Cell

Article Polo Kinase Regulates Mitotic Chromosome Condensation by Hyperactivation of Condensin DNA Supercoiling Activity Julie St-Pierre,1,2,3 Me´lanie Douziech,1,2,3 Franck Bazile,1,2,3 Mirela Pascariu,1,2 E´ric Bonneil,1 Ve´ronique Sauve´,1,2 Hery Ratsima,1,2 and Damien D’Amours1,2,* 1Institute

for Research in Immunology and Cancer de Pathologie et Biologie Cellulaire Universite´ de Montre´al, P.O. Box 6128, Station Centre-Ville, Montreal, Quebec H3C 3J7, Canada 3These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.molcel.2009.04.013 2De ´ partement

SUMMARY

A defining feature of mitosis is the reorganization of chromosomes into highly condensed structures capable of withstanding separation and large-scale intracellular movements. This reorganization is promoted by condensin, an evolutionarily conserved multisubunit ATPase. Here we show, using budding yeast, that condensin is regulated by phosphorylation specifically in anaphase. This phosphorylation depends on several mitotic regulators, and the ultimate effector is the Polo kinase Cdc5. We demonstrate that Cdc5 directly phosphorylates all three regulatory subunits of the condensin complex in vivo and that this causes a hyperactivation of condensin DNA supercoiling activity. Strikingly, abrogation of condensin phosphorylation is incompatible with viability, and cells expressing condensin mutants that have a reduced ability to be phosphorylated in vivo are defective in anaphase-specific chromosome condensation. Our results reveal the existence of a regulatory mechanism essential for the promotion of genome integrity through the stimulation of chromosome condensation in late mitosis.

INTRODUCTION The main objective of cell division is to produce two genetically identical daughter cells from a single progenitor cell. To achieve this, cells must follow a stereotypical series of carefully choreographed subcellular changes globally known as mitosis. In his seminal description of mitosis more than 100 years ago, Walther Flemming found that a central feature of this process is the condensation of chromatin fibers into microscopically visible chromosomes and the partition of those chromosomes—in the form of sister chromatids—to opposite poles of the cell (Paweletz, 2001). The fundamental importance of these events for mitosis has been confirmed since then and we now know that

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failure to efficiently reorganize chromosome structure during mitosis leads to disease or lethality. Research in the last several years has demonstrated that chromosome condensation serves multiple functions in mitosis (reviewed in Hirano, 2005; Swedlow and Hirano, 2003). First, it is required to reduce the length of chromosome arms so that they are not cut out from the rest of the chromosome during cytokinesis (Saka et al., 1994). Second, it provides structural rigidity so that chromosomes can withstand spindle-pulling forces and be moved to opposite poles of the cell (Gerlich et al., 2006; Hudson et al., 2003; Ono et al., 2003). Finally, chromosome condensation is believed to be essential to disentangle DNA replication-induced sister chromatid catenates by favoring the decatenation activity of DNA topoisomerases (Uemura et al., 1987; Wang, 1996). All those functions are independent prerequisites for efficient chromosome segregation in mitosis. Recent genetic and biochemical studies have led to the identification of several factors responsible for chromosome condensation. Chief among those factors is condensin, an 650 kDa pentameric complex playing a central role in the large-scale chromatin reorganization that gives rise to well-defined metaphase chromosomes (reviewed in Hirano, 2005). Other chromatin proteins, notably histones and topoisomerase II, play prominent roles in this process as well. These proteins act at least in part by providing the structural framework that underlies the complex and highly ordered chromatin network of mitotic chromosomes (Maeshima and Laemmli, 2003). Topoisomerase II also acts by decatenating DNA duplexes from different sister chromatids to allow chromosome arm resolution and complete chromatin condensation (Uemura et al., 1987). How condensin mediates chromosome condensation, however, remains a key unanswered question to this day. It has been proposed that the ability of this complex to reconfigure the topology of DNA strands could drive chromosome condensation by inducing the formation of large chromatin loops on mitotic chromosomes (reviewed in Swedlow and Hirano, 2003). Condensin may also create chromatin loops by tethering the base of chromatin domains onto chromosome scaffolds or by acting as a scaffold itself (Maeshima and Laemmli, 2003; Stray et al., 2005). These models are not mutually exclusive and other modes of action are clearly possible. Another key unanswered

Molecular Cell Regulation of Chromosome Condensation by Cdc5

Figure 1. Phosphorylation of Condensin in Anaphase (A–C) D506 (BRN1-3HA cdc15-2), D517 (YCG13HA cdc15-2), and D491 (YCS4-13MYC cdc15-2) cells were arrested in G1 in YEPD medium containing a factor (5 mg/ml) followed by a release into fresh medium at 37 C. Samples were taken at indicated time points and proteins were processed for western blot analysis following standard (top panels) or continuous (bottom panels) SDS-PAGE. (D–F) The percentage of budded cells (D), cells with metaphase spindles (E), and anaphase/telophase spindles (F) was determined at the indicated times. (G–I) Condensin subunits were immunoprecipitated from anaphase-arrested cells (D506, BRN13HA cdc15-2; D930, YCG1-3FLAG cdc15-2; D491, YCS4-13MYC cdc15-2) and processed for western analysis or dephosphorylation by l phosphatase.

question is how the condensin complex might be activated at the proper time in mitosis to ensure precise coordination of chromosome condensation with other key mitotic events. Phosphorylation appears to play a major role in this process since several condensin subunits are targets of mitotic kinases, notably aurora B (Lavoie et al., 2004; Lipp et al., 2007; Takemoto et al., 2007) and Cdk1 (Kimura et al., 2001, 1998; Sutani et al., 1999). At least in vitro, phosphorylation of condensin by Cdk1 activates its DNA supercoiling activity (Kimura et al., 2001, 1998), but how this might cause chromosome condensation at the molecular level remains to be established. Further, whether aurora B kinase directly phosphorylates condensin in vivo or acts indirectly to promote the phosphorylation of condensin is currently unknown. In the present study, we have investigated the nature and consequences of condensin posttranslational modifications in mitosis. We demonstrate herein that multiple subunits of the condensin complex are phosphorylated in mitosis by the Polo kinase Cdc5 and that efficient phosphorylation of condensin is required to promote the formation of normal mitotic chromosomes in vivo. RESULTS Condensin Regulatory Subunits Are Phosphorylated in Anaphase To understand how condensin is regulated in mitosis, we monitored the phosphorylation status of its subunits during the cell cycle. We focused on the three non-SMC subunits of condensin—Brn1, Ycg1, and Ycs4—because these are believed to carry the regulatory functions of the condensin complex (Kimura et al., 1998). Our approach to uncover phosphorylation is based on the fact that phosphorylation of many proteins is associated with changes in their electrophoretic migration during SDS-PAGE. Cells carrying epitope-tagged versions of Brn1, Ycg1, and Ycs4 proteins were first synchronized in G1 using mating pheromone, then released from the arrest in fresh medium at 37 C, and

samples were collected at various intervals. This experiment was carried out in a cdc15-2 mutant strain background to allow analysis of protein phosphorylation in a single cell cycle window (i.e., from G1 to telophase, the point of arrest of cdc15-2 mutants). Figures 1A–1C (top panels) show that, when using standard SDS-PAGE conditions, a small mitosis-specific gel retardation can be observed for Ycg1 but no retardation is observed for the other condensin subunits. The gel retardation seen with Ycg1 under these conditions is very similar to the phosphorylationinduced retardation observed by Lavoie et al. (2004). However, to further investigate whether Brn1 and Ycs4 are phosphorylated in mitosis, we used a modified SDS-PAGE procedure making use of alternative acrylamide mixtures (see Experimental Procedures for details). Using this alternative approach, we observed striking changes in the electrophoretic behavior of all the regulatory subunits (Figures 1A–1C, bottom panels). In particular, Brn1 and Ycg1 showed major retarded species, while a noticeable but more modest retardation was seen with Ycs4 (Figures 1A– 1C, bottom panels). In all cases, the appearance of these slowermigrating bands was concomitant with the appearance of long spindles (90 min postrelease; Figures 1D–1F), indicating that the changes we observed occurred either during or after the metaphase-anaphase transition. We next immunopurified condensin subunits from anaphasearrested cells and subjected them to phosphatase treatment. All slower-migrating bands were eliminated after phosphatase treatment (Figures 1G–1I). These observations demonstrate that the retarded bands represent phosphorylated species of condensin subunits. Phosphorylation of Condensin Depends on Polo/Cdc5 Kinase Next, we sought to identify the kinase that phosphorylates condensin subunits. The tight temporal coordination in the phosphorylation of Ycg1, Ycs4, and Brn1 during late mitosis suggests that these events might be controlled by a single kinase.

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Molecular Cell Regulation of Chromosome Condensation by Cdc5

Figure 2. Phosphorylation of Condensin Depends on Polo/Cdc5 Kinase Cells were arrested in G1 in YEPD medium with a factor followed by a release into fresh medium at 37 C. Samples were taken at indicated time points and processed for western blot analysis (left) and for budding index (right). (A) D506 (BRN1-3HA cdc15-2) and D845 (BRN1-3HA cdc5-99). (B) D517 (YCG1-3HA cdc15-2) and D848 (YCG1-3HA cdc5-99). (C) D491 (YCS4-13MYC cdc15-2) and D1204 (YCS4-13MYC cdc5-99).

Although Cdk1-cyclin B is a major mitotic kinase, it is an unlikely kinase for all the non-SMC subunits of condensin since Brn1 does not contain any serine or threonine in the minimal Cdk1 phosphorylation consensus context (i.e., ‘‘S/T-P’’) (Ubersax et al., 2003). Instead, the yeast Polo kinase Cdc5 is a good candidate as it is a major cell cycle kinase that is involved in multiple aspects of chromosome behavior during mitosis (Petronczki et al., 2008). We constructed cdc5 mutant strains carrying epitope-tagged condensin subunits and tested the involvement of Cdc5 in their phosphorylation during the cell cycle. Strikingly, all phosphorylation-induced gel retardation of condensin regulatory subunits was lost in cells lacking Cdc5 activity (Figures 2A– 2C). We tested several cdc5 mutant alleles and all gave rise to complete abrogation of condensin phosphorylation by gel retardation assay (data not shown). Taken together, these results show that condensin phosphorylation during mitosis depends on Cdc5 activity. Phosphorylation of Condensin Depends Only Partially on Ipl1 The results obtained above argue that most, if not all, phosphorylation of condensin non-SMC subunits is mediated by Cdc5. However, Ipl1 kinase, the yeast homolog of vertebrate aurora B, has previously been involved in the phosphorylation of Ycg1

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(Lavoie et al., 2004). To test whether Ipl1 also regulates condensin phosphorylation under our conditions, we constructed yeast strains bearing conditional alleles of ipl1 and tested the phosphorylation of epitope-tagged Brn1, Ycg1, and Ycs4 in these mutant cells. As can be seen in Figure 3A, there is a delay and reduction in the kinetics of Ycg1 phosphorylation in ipl1-2 mutants. The reduction was most obvious when comparing the ratio of phosphorylated versus nonphosphorylated bands at the 110–130 min time points in control and mutant cells (Figure 3A). Interestingly, however, phosphorylation of Ycg1 was not completely lost in the absence of Ipl1 activity. Similar results were obtained for Brn1 and Ycs4 in ipl1 mutant cells (Figures 3B and 3C and Figure S1 available online). These results are consistent with those of Lavoie et al. (2004) who observed that Ipl1 was required for maximal phosphorylation of Ycg1, but that some phosphorylation remained in ipl1 mutants. Cdc5 Is Necessary and Sufficient to Induce Condensin Phosphorylation In Vivo and In Vitro We next wanted to investigate whether Cdc5 is sufficient to induce condensin phosphorylation at a stage of the cell cycle when this event does not normally occur (i.e., S phase). Strains carrying epitope-tagged versions of condensin subunits were transformed with plasmids containing GAL10-1 promoter-driven

Molecular Cell Regulation of Chromosome Condensation by Cdc5

Figure 3. Condensin Phosphorylation Is Partially Dependent on Aurora B/Ipl1 Kinase Cells were arrested in G1 in YEPD medium with a factor followed by a release into fresh medium at 37 C. Samples were taken at indicated time points and processed for western blot analysis (left) and for budding index (right). (A) D517 (YCG1-3HA cdc15-2) and D688 (YCG1-3HA ipl1-2 cdc15-2). (B) D506 (BRN1-3HA cdc15-2) and D656 (BRN1-3HA ipl1-182 cdc15-2). (C) D491 (YCS4-13MYC cdc15-2) and D1229 (YCS4-13MYC ipl1-2 cdc15-2).

CDC5 or IPL1. These strains were first arrested in S phase for 2 hr with hydroxyurea (HU) and galactose was subsequently added to the culture medium to induce expression of the kinases (in the presence of HU). As can be seen in Figure 4A, control cells arrested in S phase with HU did not exhibit condensin phosphorylation throughout the experimental time frame (60 min). However, induction of CDC5 expression led to robust phosphorylation of Brn1, Ycg1, and Ycs4 subunits as early as 30 min postinduction and to maximal levels after 1 hr (Figure 4A). In contrast, IPL1 overexpression did not lead to increased phosphorylation of condensin subunits. We conclude from these experiments that CDC5 is both necessary and sufficient to induce condensin phosphorylation. We then sought to determine whether Cdc5 has the ability to directly phosphorylate condensin in vitro. To answer this question, condensin was purified from yeast lysates and was used as substrate in kinase reactions using radiolabeled ATP and purified Cdc5, Ipl1-Sli15, or human Cdk1-cyclin B (along with generic substrates). Cdk1 kinase was used as a positive control since yeast Smc4 is a known substrate of Cdk1 in vitro (Ubersax et al., 2003). Once the phosphorylation reactions were completed, proteins were separated on standard SDS-polyacrylamide gels to allow identification of the radiolabeled bands. As can be seen in Figure 4B, Cdc5 could efficiently phosphorylate condensin subunits in vitro, as demonstrated by the appearance

of 32P-radiolabeled bands corresponding to the molecular weight of condensin subunits. It is apparent from this experiment that Brn1, Ycg1, and Smc4 are directly phosphorylated by Cdc5 (Figure 4B). We also observed a signal at the position of Ycs4 (133 kDa) and Smc2 (134 kDa) but we cannot ascertain whether only one or both proteins are phosphorylated by Cdc5 since these proteins cannot be separated on gel (Freeman et al., 2000). Strikingly, although the Ipl1-Sli15 complex could efficiently phosphorylate itself (Figure 4B, band marked with asterisk) and a generic substrate (MBP; bottom panel), it was unable to phosphorylate any subunit of the condensin complex. As expected, active Cdk1 could efficiently phosphorylate Smc4 as well as some of the regulatory subunits to a much lesser extent (Figure 4B). The results presented in this section, together with our genetic analyses, strongly suggest that Cdc5 is the kinase that phosphorylates condensin in vivo. Identification of the Phosphorylation Sites on Condensin What could be the impact of the anaphase-specific phosphorylation of condensin? To answer this question, we set out to identify phosphorylated sites on condensin by mass spectrometry. Condensin subunits were immunopurified from lysates of anaphase-arrested cells and separated by electrophoresis, and their corresponding bands were analyzed by nano-LC-MS/MS mass spectrometry.

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Molecular Cell Regulation of Chromosome Condensation by Cdc5

Figure 4. Cdc5 Is a Condensin Kinase (A) D506 (BRN1-3HA cdc15-2), D517 (YCG1-3HA cdc15-2), and D491 (YCS413MYC cdc15-2) cells carrying an empty multicopy vector or vectors for overexpression of CDC5 or IPL1 were arrested in S phase in YEP-raffinose medium containing 200 mM HU. GAL-CDC5 or GAL-IPL1 expression was induced by addition of galactose to the culture medium. Samples were taken at indicated time points and proteins were processed for western blot analysis for condensin subunits (top panels), Cdc5-StrepTagII (middle panels), and Ipl1-HA (bottom panels). (B) Purified condensin was incubated alone or with purified Cdc5, Ipl1-Sli15 complex, or Cdk1-cyclin B complex. Phosphorylation reactions were carried out in the presence of 32P-ATP and generic phosphorylation substrates (Histone H1, Casein, and MBP). After the reaction, proteins were separated by standard SDS-PAGE. The position of proteins is shown on the Coomassie-stained gels on the left, while autoradiographs of these gels are shown on the right. Top panels are 4%–12% gradient gels (to visualize condensin subunits), whereas bottom panels are 12.5% gels (to visualize generic phosphorylation substrates). Asterisks indicate the position of the kinases (or their regulatory subunits), whereas bullets indicate the position of all condensin subunits.

Several phosphorylated sites on Brn1, Ycg1, and Ycs4 were identified by this approach (Figures 5A–5D). Thirty-six individual sites were unambiguously confirmed by manual inspection of their mass spectra (Figures 5A–5D and S2–S4). Sites were more or less evenly distributed between Brn1 (15 sites), Ycg1 (13 sites confirmed; 1 ambiguous), and Ycs4 (7 sites; Figures 5A–5D). In addition to 31 sites previously unreported, we identified all the sites that were previously detected in proteomewide screens (except phospho-Ser933 in Ycg1; Chi et al., 2007; Smolka et al., 2007). Interestingly, a significant fraction of the identified sites conform to the recently defined consensus

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sequence for phosphorylation by Cdc5 (Brar et al., 2006) or Plk1 (Nakajima et al., 2003). Furthermore, a striking characteristic of condensin’s phosphorylation sites is their strong tendency to cluster in relatively short (40–100 aa) regions that tend to be somewhat divergent even among homologs from closely related species (Figures S5–S7). We next wished to validate the phosphorylation of some of these sites in mitosis. Antibodies to phospho-Ser256 of Brn1 (this study) and phospho-Ser933 of Ycg1 (Smolka et al., 2007) were generated and used to probe immunopurified condensin from G1 and anaphase extracts. As can be seen in Figures 5E–5G, these antibodies detected only the phosphorylationinduced slow-migrating bands of Brn1 and Ycg1. Further, mutagenesis of Ser933 to Ala in Ycg1 eliminated the signal from the phospho-Ser933 antibody (Figure 5F). Interestingly, mutation of Ser256 in Brn1 did not prevent the phospho-Ser256 antibody from recognizing Brn1 but dephosphorylation of condensin did (Figure 5G). We hypothesize that the phospho-Ser256 antibody has the ability to recognize other phosphorylation sites in Brn1 because its epitope fits a perfect Cdc5 consensus sequence (Brar et al., 2006). To further demonstrate that the sites we identified were bona fide phosphorylation sites in vivo, we constructed a series of yeast strains carrying alanine substitutions at the positions of all phosphorylation sites identified by mass spectrometry. We also included three additional mutations that fell within sequences not covered by our mass spectrometry analyses of Brn1 and/or that fitted the perfect consensus for phosphorylation by Cdc5/Plk1 (S264A, S432A, and S544A). As can be seen from Figures 5H–5J, these mutants have strong defects in anaphase-specific phosphorylation. In particular, brn1-570 and ycs4-543 mutants show no detectable phosphorylation-induced gel retardation (Figures 5H and 5I; see Experimental Procedures for the sites mutated in all phospho-alleles). The ycg1 mutant carrying most of the identified phosphorylation sites (ycg1-515) had a severe but incomplete reduction in phosphorylation (Figure 5J). This is likely due to the fact that not all phosphorylation sites were identified in our mass spectrometry analysis. We reasoned that additional sites were likely to be located in the divergent C-terminal region of Ycg1 since most sites identified so far cluster in this specific region of the protein (Figure 5A). We therefore created another mutant, ycg1-521, in which all putative Cdc5-dependent sites remaining in the C-terminal tail of Ycg1 were removed from the protein. Strikingly, phosphorylation of Ycg1 was almost completely eliminated in this mutant (Figure 5J). To further demonstrate that the sites identified by mass spectrometry were genuine Cdc5 phosphorylation sites, we overexpressed CDC5 in HU-arrested cells bearing phospho mutant alleles of condensin. Both wild-type Ycg1 and Ycs4 were efficiently phosphorylated upon CDC5 overexpression, whereas the phospho mutant versions of these proteins showed severely blunted phosphorylation-induced gel shifts (Figure 5K). Interestingly, we repeatedly observed that cells carrying the brn1-570 mutant allele did not overexpress CDC5 under these conditions (Figure 5K). Additional experiments showed that this allele completely abrogates gene expression from the GAL1 or GAL10 promoters (Figure S8), thereby preventing us from analyzing

Molecular Cell Regulation of Chromosome Condensation by Cdc5

Figure 5. Identification of Phosphorylated Residues on Condensin Subunits (A) Schematic representation of Brn1, Ycg1, and Ycs4. The position of each phosphorylated residue identified by mass spectrometry is marked by a bar and residue number above and below the proteins. Protein segments highlighted in green represent regions enriched in phosphorylation sites. Note that phospho-Ser933 of Ycg1 was found by mass spectrometry by Smolka et al. (2007) and confirmed herein using an anti-phospho-Ser933 antibody (see below). Proteins and phospho-residue positions are drawn to scale. (B–D) Sequence context of the phosphorylated residues in Brn1, Ycg1, and Ycs4. Phosphorylated residues that fit the consensus for Cdc5 or Plk1 are marked with an asterisk (Brar et al., 2006; Nakajima et al., 2003). Note the enrichment for hydrophobic amino acids (highlighted in yellow) following the phosphorylated sites (in bold and highlighted in gray) and the tendency of phosphorylated sites to cluster together (underlined and highlighted in gray). (E) Lysates from D517 (YCG1-3HA cdc15-2) were prepared from G1-arrested (0 min) and anaphase-arrested (120 min) cells. Ycg1-3HA was immunoprecipitated from these lysates and processed for western analysis using anti-HA or anti-phospho-Ser933 antibodies. (F) Lysates from D517 (YCG1-3HA cdc15-2) and D897 (ycg1-S933A cdc15-2) were processed as above for western analysis using anti-HA or anti-phospho-Ser933 antibodies. (G) Lysates from D506 (BRN1-3HA cdc15-2) and D946 (brn1-S256A cdc15-2) were prepared from G1-arrested (0 min) and anaphase-arrested (120 min) cells. Brn1-3HA was immunoprecipitated from these lysates and processed for western analysis using anti-HA or anti-phospho-Ser256 antibodies. An additional dephosphorylation control was performed with l phosphatase. (H–J) Cells were arrested in G1 in YEPD medium with a factor followed by a release into fresh medium at 37 C. Samples were taken at the indicated time points and processed for western blot analysis (left) and for budding index (right). (H) D506 (BRN1-3HA cdc15-2) and D1231 (brn1-570-3HA cdc15-2). (I) D491 (YCS4-13MYC cdc15-2) and D1165 (ycs4-54313MYC cdc15-2). (J) D517 (YCG1-3HA cdc15-2), D1135 (ycg1-515-3HA cdc15-2), and D1139 (ycg1-521-3HA cdc15-2). Note that pictures for wild-type Ycg1 and its phospho mutant alleles are different exposures of the same gel. (K) D517 (YCG1-3HA cdc15-2) and D1139 (ycg1-521-3HA cdc15-2), D506 (BRN1-3HA cdc15-2) and D1231 (brn1570-3HA cdc15-2), and D491 (YCS4-13MYC cdc15-2) and D1165 (ycs4-543-13MYC cdc15-2) cells carrying CDC5 overexpression vectors were arrested in S phase and CDC5 expression was induced for 60 min as before. Top panels show condensin subunits and bottom panels show Cdc5-StrepTagII. W and M refer to wild-type and phospho mutant condensins, respectively, whereas asterisks marks the lane where Cdc5 is not expressed at 60 min. (L) Wild-type and phospho mutant condensin complexes phosphorylated with purified Cdc5 in the presence of 32 P-ATP (left, Coomassie-stained gel; right, autoradiogram). Reduction in 32P signal in non-SMC subunits must be considered relative to Smc4 signal (positive control between lanes). W and M refer to wild-type and phospho mutant condensins, respectively.

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Figure 6. Phenotype of Condensin Phosphorylation Mutants (A) Ten-fold dilution series of individual phospho mutants of condensin subunits were spotted on YEPD plates to evaluate cell growth. D506 (BRN1-3HA cdc15-2), D1231 (brn1-5703HA cdc15-2), D491 (YCS4-13MYC cdc15-2), D1165 (ycs4567-13MYC cdc15-2), D517 (YCG1-3HA cdc15-2), D1135 (ycg1-515-3HA cdc15-2), D1138 (ycg1-520-3HA cdc15-2), D1139 (ycg1-521-3HA cdc15-2), and D1088 (ycg1-912-3HA cdc15-2). The number of phosphorylation sites mutated to alanine in each mutant is shown on the right. (B) A heterozygous diploid strain carrying brn1-570::URA3 and ycg1-488::kanMX6 alleles was induced to sporulate and tetrads were dissected on YEPD plates (left panel). The genotype of viable segregants was scored on synthetic medium lacking uracil (middle panel) and rich medium containing G418 (right panel). (C) D1237 (BRN1-3HA YCG1-3HA YCS4-13MYC cdc15-2) and D1225 (brn1-539-3HA ycg1-488-3HA ycs4-543-13MYC cdc15-2) cells were arrested in anaphase at 37 C and processed for visualization of the rDNA array on chromosome XII by FISH. The morphology of the rDNA array was scored in >300 cells, and the actual percentage of cells in each category is shown within the pie charts. (D) Micrographs showing the morphology of the rDNA array in condensin phospho mutants and cdc15-2 cells from the experiment shown in (C). (E) D754 (cdc15-2) and D777 (cdc5-99) cells were arrested in anaphase at 37 C and processed for rDNA array visualization by FISH as above. (F) Micrographs showing the morphology of the rDNA array in cdc5-99 mutants and cdc15-2 cells from the experiment shown in (E).

Brn1-570 phosphorylation levels upon CDC5 overexpression. To bypass this problem, we performed in vitro phosphorylation reactions with purified wild-type and phospho mutant condensins. Removal of the phosphorylation sites caused a strong reduction in the ability of Cdc5 to phosphorylate the non-SMC subunits of the complex in vitro (Figure 5L), relative to the phosphorylation observed with our control substrate, Smc4. In particular, we do not detect any signal at the position of Ycs4 and the phosphorylation-induced gel shift of Ycg1 is completely abolished in the mutant complex. Further, the 32P signals from Brn1 and Ycg1 are significantly lowered in the mutant relative to the wild-type complex (Figure 5L). Taken together, the overexpression experiments and in vitro phosphorylation analyses indicate that we have identified the majority of the phosphorylation sites on condensin non-SMC subunits and that these sites are direct targets for Cdc5 phosphorylation. Lethality and rDNA Condensation Defects in Phosphorylation Site Mutants of Condensin We next sought to determine the role of condensin phosphorylation in vivo. Interestingly, yeast expressing individual phosphodeficient alleles of condensin subunits from their endogenous locus are viable and show no detectable growth defect (Figure 6A). Two possibilities could account for this. First, the phosphorylation of condensin may regulate its function without being essential for viability. Second, the phosphorylation of individual subunits may act together with the phosphorylation of other subunits to control condensin complex activity. To test

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these hypotheses, we decided to combine two phosphodeficient alleles of condensin subunits in a single yeast strain. To achieve this, we first mated individual haploid strains carrying brn1-570 and ycg1-488 and sporulated the resulting diploid strain. Strikingly, the pattern of viability following dissection of the sporulated heterozygous diploid strain suggested strongly that combining two phospho-deficient alleles of condensin subunits causes synthetic lethality in haploid segregants (Figure 6B). This conclusion was confirmed by subsequent scoring of the genetic markers associated with each allele (URA3 and kanMX6; Figure 6B). Taken together, these results indicate that artificially preventing condensin phosphorylation blocks the execution of a process essential for viability. What might be the cellular process whose defect is responsible for the lethality of condensin phosphorylation mutants? A likely cause for this phenotype is the process of chromosome condensation, as it is the main cellular function of the condensin complex. Testing this hypothesis, however, was complicated by the fact that complete removal of phosphorylation sites in more than one condensin subunit is not compatible with viability (Figure 6B). To bypass this complication, we constructed a triple condensin subunit mutant that is only partially defective in phosphorylation (i.e., brn1-539 ycg1-488 ycs4-543). This triple mutant was viable and showed only a mild growth defect. We then set out to determine the impact of reducing condensin phosphorylation on chromosome condensation during anaphase. For this, we monitored the morphology of the ribosomal DNA (rDNA) array on chromosome XII by FISH since this locus undergoes a

Molecular Cell Regulation of Chromosome Condensation by Cdc5

well-documented series of structural changes during mitosis (Guacci et al., 1994; Lavoie et al., 2004; Machin et al., 2005). In particular, anaphase is characterized by the transient appearance of a short rDNA ‘‘line’’ morphology that depends on both Cdc14 and condensin (Guacci et al., 1994; Sullivan et al., 2004). This short line phenotype is seen in about 65% of cdc15-2 cells arrested in anaphase with the rest of the population showing an amorphous ‘‘puff’’ or a dense rDNA ‘‘cluster’’ morphology (Lavoie et al., 2004; Figure 6C). Strikingly, when we analyzed the morphology of the rDNA in cdc15-2-arrested cells carrying condensin phospho mutant alleles, we consistently observed a strong reduction (4-fold compared to controls) in the proportion of cells showing the anaphase-specific rDNA line phenotype (Figures 6C and 6D). This reduction was accompanied by a corresponding increase in rDNA cluster morphology, which is not usually observed at high frequency in anaphase cells (Lavoie et al., 2004). As expected, the condensation defect in the partial phosphorylation mutant is not complete (16% remaining rDNA lines; Figure 6C), which likely explains why the mutant strain remains viable. We next wanted to determine whether Cdc5 was also required for rDNA condensation in anaphase. Consistent with its role in promoting condensin phosphorylation, mutants defective in Cdc5 showed a marked reduction in anaphase rDNA line formation and a corresponding increase in rDNA cluster morphology compared to cdc15-2 control cells (Figures 6E and 6F). This phenotype is consistent with the observation that condensin is not enriched in the nucleolus in anaphase-arrested cdc5 mutant cells (Wang et al., 2004). Taken together, our results clearly establish that Cdc5-dependent phosphorylation of condensin is essential for viability and that preventing this regulatory event causes defects in chromosome morphology during anaphase. Phosphorylation of Condensin by Cdc5 Activates Its DNA Supercoiling Activity We next wanted to investigate how Cdc5-dependent phosphorylation regulates condensin activity in vitro. To achieve this, the condensin complex was overexpressed in yeast and purified to 95% homogeneity using a combination of affinity and size exclusion chromatography (Figure 7A and Experimental Procedures). The ability of condensin to introduce positive supercoils in DNA is the hallmark activity of this enzyme in higher eukaryotes (Kimura et al., 2001; Kimura and Hirano, 1997). This prompted us to investigate whether purified yeast condensin was similar to its metazoan counterparts in its ability to supercoil DNA in vitro. As shown in Figure 7B (lane 7), yeast condensin purified from asynchronous cells has no intrinsic DNA supercoiling activity. However, prephosphorylation of yeast condensin with Cdk1cyclin B activates its DNA supercoiling activity (Figure 7B, lanes 15–20). Further, DNA supercoiling activity is only seen at high concentration of yeast condensin complex relative to DNA (Figure 7B, lanes 15–20), suggesting a cooperative mode of action similar to its metazoan counterparts (Kimura and Hirano, 1997). We next asked whether Cdc5-mediated phosphorylation affected condensin activity. As shown in Figure 7B (lane 8), phosphorylation of condensin by Cdc5 alone did not activate its DNA supercoiling activity. However, phosphorylation of condensin by both Cdk1 and Cdc5 gave rise to a striking increase in its

DNA supercoiling activity (Figure 7B, lane 9–14). The stimulation of condensin DNA supercoiling activity seen in the presence of both Cdk1 and Cdc5 phosphorylation is approximately 5-fold over the stimulation afforded by Cdk1 alone (Figure 7B, bottom). Importantly, in vitro phosphorylation experiments showed that Cdc5 kinase does not act by stimulating Cdk1 activity toward Smc4 (and vice versa) under our assay conditions (Figures 7C and 7D). Taken together, these results indicate that phosphorylation of condensin by Cdc5 hyperactivates its Cdk1-dependent DNA supercoiling activity in vitro. DISCUSSION We report here that condensin is regulated by Cdc5-mediated phosphorylation during mitosis. This phosphorylation occurs on the regulatory non-SMC subunits of the condensin complex, namely Brn1, Ycg1, and Ycs4, and appears late in mitosis when mitotic spindles start to elongate. Further, this phosphorylation increases to high levels when cells reach the final stages of mitosis. We demonstrate that phosphorylation of condensin is essential to promote efficient chromosome condensation at the rDNA in anaphase cells and that it hyperactivates condensin DNA supercoiling activity in vitro. Taken together, our results reveal the existence of a regulatory mechanism essential for the promotion of genome integrity through the stimulation of chromosome condensation in late mitosis. Activation of Condensin during Mitosis Requires Phosphorylation A major question regarding the mode of action of condensin is how it becomes competent to mediate chromosome condensation specifically during mitosis. Elegant biochemical work using Xenopus laevis mitotic extracts provided strong evidence for a fundamental role of mitotic kinases in the activation of condensin. In particular, Hirano and collaborators showed that condensin purified from mitotic extracts can condense chromosomes in vitro and that this activity correlates with condensin’s ability to introduce supercoils in DNA in vitro (Hirano et al., 1997; Kimura and Hirano, 1997). Importantly, the authors showed that direct phosphorylation of human and Xenopus condensins by Cdk1 is required to activate their DNA supercoiling activity in vitro (Kimura et al., 2001, 1998). Is Cdk1 phosphorylation sufficient to render condensin fully competent to induce chromosome condensation? We demonstrate in this study that phosphorylation of yeast condensin only on Cdk1 sites is insufficient to promote normal rDNA condensation in vivo. Interestingly, there is strong evidence in frog extracts that kinases in addition to Cdk1 are required for full mitotic phosphorylation of condensin (Kimura et al., 1998). Consistent with this, proteome-wide mass spectrometry analyses have revealed the existence of 40 phosphorylation sites on the non-SMC subunits of mammalian condensin (Figure S9). About 80% of the sites identified in these analyses are non-CDK sites, suggesting that kinases other than CDK are major regulators of condensin in mammalian cells. It is important to note that there appear to be some speciesspecific differences in the phosphorylation of condensin. For instance, several phosphorylation sites identified in metazoan condensin tend to cluster in N- and C-terminal extensions that

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Figure 7. Phosphorylation of Condensin by Cdc5 Hyperactivates Its DNA Supercoiling Activity (A) Purification of condensin from yeast. Condensin holoenzyme was purified from crude extract by nickel-NTA and Streptactin affinity chromatography followed by size exclusion on Superose 6. The elution profile of condensin from the gel filtration column is shown on the top graph, whereas the purity of condensin at each purification step is shown in the Coomassie-stained gel below the graph. Fractions used for DNA supercoiling assays are marked. (B) DNA supercoiling activity of yeast condensin. Purified condensin holoenzyme was first preincubated with ATP and either Cdk1 alone (lanes 15–20) or Cdc5 plus Cdk1 (lanes 9–14) for 40 min to induce phosphorylation. Then, phosphorylated condensin was incubated with relaxed plasmid DNA to allow the introduction of positive DNA supercoils in the plasmid. Plasmid topoisomers were resolved on agarose gels containing chloroquine. The graph below the gel shows the supercoiling activity in lanes 9–20 as the ratio of supercoiled DNA over relaxed DNA in each lane. Fold increase is relative to the basal condensin activity seen at low condensin concentration in the presence of Cdk1 (i.e., activity in lane 15). (C) Cdc5 does not stimulate phosphorylation of condensin by Cdk1. Purified condensin was phosphorylated with purified Cdc5 alone, Cdk1 alone, or both kinases together in the presence of 32P-ATP (left, Coomassie-stained gel; right, autoradiogram). Asterisks indicate the position of Cdc5 or cyclin B, whereas bullets indicate the position of all condensin subunits. (D) Quantitation of the results shown in (C). Results are normalized to the signal obtained with Cdc5 alone. Note that the activity of Cdc5 toward Brn1, Ycg1, and Ycs4 is slightly reduced in the presence of Cdk1 but that the vast majority of Cdc5 kinase activity (80%) remains present under this condition.

are absent in yeast (i.e., see schematic of CapH and CapD2 in Figure S9). In the case of hCapD2, part of its C-terminal extension has been implicated in chromosome targeting (Ball et al., 2002), a function that may not be conserved in yeast due to constitutive nuclear/chromatin localization of condensin in this organism (Freeman et al., 2000). It is conceivable that the difference between yeast and metazoan condensin phosphorylation may also reflect different timing in the initiation of chromosome condensation in these organisms (human condensin I and II are activated in prometaphase and prophase, respectively, whereas yeast condensin acts in or close to metaphase; Guacci et al., 1994; for review see Hirano, 2005). Although specific phosphorylation sites and their usage may differ between organisms, it is clear that both yeast and metazoan condensins require Cdk1 phosphorylation to become active in vitro. The notion that condensins are also activated by Cdk1 phosphorylation in vivo is highly probable but remains to be proven. Nonetheless, given the very

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high abundance of non-Cdk phosphorylation sites identified in human condensin (see Figure S9), it seems likely that mitotic kinases distinct from Cdk1 will also regulate condensin in higher eukaryotes. Whether these kinases include mammalian Polo family members like in yeast remains to be tested experimentally. Dual Regulation of Condensin by Polo/Cdc5 and Cdk1 In Vivo How might phosphorylation of condensin by Cdc5 regulate its activity in vivo? It is clear from our in vitro experiments that the phosphorylation of condensin with Cdc5 alone is insufficient to activate its DNA supercoiling activity. This suggests that Cdc5mediated phosphorylation must act by adding another level of activation (i.e., hyperactivation) to condensin over the level achieved by Cdk1-mediated phosphorylation alone. We envision at least two reasons why there is a need to hyperactivate condensin during mitosis. First, the activation of

Molecular Cell Regulation of Chromosome Condensation by Cdc5

condensin by Cdc5 could functionally compensate for the lowering of Cdk1 activity experienced by anaphase/telophase cells relative to metaphase cells (Baumer et al., 2000; Yeong et al., 2000). This might be important to ensure that chromosomes do not start to decondense before their segregation is completed at the end of anaphase. This hypothesis is consistent with the fact that chromosome condensation is maintained during anaphase in mammalian cells (Mora-Bermudez et al., 2007) and at the rDNA locus in yeast (Guacci et al., 1994). Second, phosphorylation of condensin by Cdc5 might alter its charge distribution and render it competent for a specialized function in late mitosis. It is noteworthy that Cdc5 tends to phosphorylate several of its substrates on multiple residues in vitro and in vivo. Such substrates include Swe1 (16 sites; 819 residues; Sakchaisri et al., 2004), Cfi1/Net1 (17 sites; 1189 residues; Loughrey Chen et al., 2002), Bfa1 (11 sites; 574 residues; Hu et al., 2001), and Rec8 (11 sites; 680 residues; Brar et al., 2006). In most of these cases, removal of all the phosphorylation sites was required to demonstrate the role of Cdc5-dependent phospho-regulation on the target substrates (Brar et al., 2006; Hu et al., 2001; Loughrey Chen et al., 2002; Sakchaisri et al., 2004). This situation is similar to what we have observed with condensin, both in terms of the density of phosphorylation sites identified on each subunit and the need to remove all the sites to uncover the function of phosphorylation. The high abundance of phosphorylation sites identified in the non-SMC subunits of mammalian condensin (Figure S9) indicates that regulation by multisite phosphorylation might be a conserved strategy with similar mechanistic consequences for condensins from multiple species. This hypothesis is admittedly speculative but represents a simple and appealing explanation for the current observations.

and buffer system according to the manufacturer’s instructions (Amresco). Standard procedures with minor modifications were used for protein purification (Stray et al., 2005), LC-MS/MS mass spectrometry analyses (Brar et al., 2006), kinase assays (Hu et al., 2001), and DNA supercoiling assays (Kimura and Hirano, 1997; Stray et al., 2005). Detailed description of all experimental procedures is included in the Supplemental Data.

Chromosome Condensation and Segregation Defects We report here that phosphorylation of condensin is highly regulated in vivo and is required to maintain chromosome morphology during cell division. It is noteworthy that inactivation of several factors regulating condensin phosphorylation causes striking defects in chromosome segregation (D’Amours et al., 2004; Machin et al., 2005; Sullivan et al., 2004; Torres-Rosell et al., 2004; Wang et al., 2004) and that these defects may impact the maintenance of chromosome condensation during anaphase. It will be interesting to investigate potential connections between these events and checkpoint mechanisms such as the NoCut pathway (Norden et al., 2006). Furthermore, given the importance of chromosome segregation defects in the generation of aneuploidy, it appears likely that abrogating anaphase-specific condensation might have important consequences on genome integrity. In this regard, it will be important to test whether Plk1 is required for anaphase condensation in human cells and whether defects in this process can lead to tumorigenesis.

Baumer, M., Braus, G.H., and Irniger, S. (2000). Two different modes of cyclin clb2 proteolysis during mitosis in Saccharomyces cerevisiae. FEBS Lett. 468, 142–148.

EXPERIMENTAL PROCEDURES All yeast strains are isogenic with strain W303. Standard procedures were used for yeast culture, genetics, immunofluorescence, and protein extract preparation (D’Amours et al., 2004 and references therein). Phosphorylationdependent gel-retardation assays were performed using Next Gel acrylamide

SUPPLEMENTAL DATA The Supplemental Data include Supplemental Experimental Procedures and nine figures and can be found with this article online at http://www.cell.com/ molecular-cell/supplemental/S1097-2765(09)00242-1. ACKNOWLEDGMENTS We are grateful to Alain Verreault and David Morgan for helpful discussions. We thank Todd Stukenburg for providing plasmids, Brad Cairns and Sue Biggins for yeast strains, James Fethie`re for the purified Ipl1-Sli15, and members of the D’Amours laboratory for their critical reading of the manuscript. M.D. and F.B are supported by postdoctoral fellowships from the Canadian Institute for Health Research (CIHR) and La Fondation Recherche Medicale, respectively. J.S.-P. was supported by CIHR and Cole Foundation postdoctoral fellowships. This research was supported by a grant to D.D. from the Terry Fox Foundation through the National Cancer Institute of Canada (#017277). D.D. holds a Canada Research Chair in Cell Cycle Regulation and Genomic Integrity. Received: December 16, 2008 Revised: March 23, 2009 Accepted: April 10, 2009 Published: May 28, 2009 REFERENCES Ball, A.R., Schmiesing, J.A., Zhou, C., Gregson, H.C., Okada, Y., Doi, T., and Yokomori, K. (2002). Identification of a chromosome-targeting domain in the human condensin subunit CNAP1/hCAP-D2/Eg7. Mol. Cell. Biol. 22, 5769– 5781.

Brar, G.A., Kiburz, B.M., Zhang, Y., Kim, J.E., White, F., and Amon, A. (2006). Rec8 phosphorylation and recombination promote the step-wise loss of cohesins in meiosis. Nature 441, 532–536. Chi, A., Huttenhower, C., Geer, L.Y., Coon, J.J., Syka, J.E., Bai, D.L., Shabanowitz, J., Burke, D.J., Troyanskaya, O.G., and Hunt, D.F. (2007). Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proc. Natl. Acad. Sci. USA 104, 2193–2198. D’Amours, D., Stegmeier, F., and Amon, A. (2004). Cdc14 and condensin control the dissolution of cohesin-independent chromosome linkages at repeated DNA. Cell 117, 455–469. Freeman, L., Aragon-Alcaide, L., and Strunnikov, A. (2000). The condensin complex governs chromosome condensation and mitotic transmission of rDNA. J. Cell Biol. 149, 811–824. Gerlich, D., Hirota, T., Koch, B., Peters, J.M., and Ellenberg, J. (2006). Condensin I stabilizes chromosomes mechanically through a dynamic interaction in live cells. Curr. Biol. 16, 333–344. Guacci, V., Hogan, E., and Koshland, D. (1994). Chromosome condensation and sister chromatid pairing in budding yeast. J. Cell Biol. 125, 517–530. Hirano, T. (2005). Condensins: organizing and segregating the genome. Curr. Biol. 15, R265–R275. Hirano, T., Kobayashi, R., and Hirano, M. (1997). Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein. Cell 89, 511–521.

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Hu, F., Wang, Y., Liu, D., Li, Y., Qin, J., and Elledge, S.J. (2001). Regulation of the Bub2/Bfa1 GAP complex by Cdc5 and cell cycle checkpoints. Cell 107, 655–665. Hudson, D.F., Vagnarelli, P., Gassmann, R., and Earnshaw, W.C. (2003). Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes. Dev. Cell 5, 323–336. Kimura, K., and Hirano, T. (1997). ATP-dependent positive supercoiling of DNA by 13S condensin: a biochemical implication for chromosome condensation. Cell 90, 625–634. Kimura, K., Hirano, M., Kobayashi, R., and Hirano, T. (1998). Phosphorylation and activation of 13S condensin by Cdc2 in vitro. Science 282, 487–490. Kimura, K., Cuvier, O., and Hirano, T. (2001). Chromosome condensation by a human condensin complex in Xenopus egg extracts. J. Biol. Chem. 276, 5417–5420. Lavoie, B.D., Hogan, E., and Koshland, D. (2004). In vivo requirements for rDNA chromosome condensation reveal two cell-cycle-regulated pathways for mitotic chromosome folding. Genes Dev. 18, 76–87. Lipp, J.J., Hirota, T., Poser, I., and Peters, J.M. (2007). Aurora B controls the association of condensin I but not condensin II with mitotic chromosomes. J. Cell Sci. 120, 1245–1255. Loughrey Chen, S., Huddleston, M.J., Shou, W., Deshaies, R.J., Annan, R.S., and Carr, S.A. (2002). Mass spectrometry-based methods for phosphorylation site mapping of hyperphosphorylated proteins applied to Net1, a regulator of exit from mitosis in yeast. Mol. Cell. Proteomics 1, 186–196. Machin, F., Torres-Rosell, J., Jarmuz, A., and Aragon, L. (2005). Spindle-independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase. J. Cell Biol. 168, 209–219. Maeshima, K., and Laemmli, U.K. (2003). A two-step scaffolding model for mitotic chromosome assembly. Dev. Cell 4, 467–480. Mora-Bermudez, F., Gerlich, D., and Ellenberg, J. (2007). Maximal chromosome compaction occurs by axial shortening in anaphase and depends on Aurora kinase. Nat. Cell Biol. 9, 822–831. Nakajima, H., Toyoshima-Morimoto, F., Taniguchi, E., and Nishida, E. (2003). Identification of a consensus motif for Plk (Polo-like kinase) phosphorylation reveals Myt1 as a Plk1 substrate. J. Biol. Chem. 278, 25277–25280. Norden, C., Mendoza, M., Dobbelaere, J., Kotwaliwale, C.V., Biggins, S., and Barral, Y. (2006). The NoCut pathway links completion of cytokinesis to spindle midzone function to prevent chromosome breakage. Cell 125, 85–98. Ono, T., Losada, A., Hirano, M., Myers, M.P., Neuwald, A.F., and Hirano, T. (2003). Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell 115, 109–121. Paweletz, N. (2001). Walther Flemming: pioneer of mitosis research. Nat. Rev. Mol. Cell Biol. 2, 72–75. Petronczki, M., Lenart, P., and Peters, J.M. (2008). Polo on the rise—from mitotic entry to cytokinesis with Plk1. Dev. Cell 14, 646–659.

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Saka, Y., Sutani, T., Yamashita, Y., Saitoh, S., Takeuchi, M., Nakaseko, Y., and Yanagida, M. (1994). Fission yeast cut3 and cut14, members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis. EMBO J. 13, 4938–4952. Sakchaisri, K., Asano, S., Yu, L.R., Shulewitz, M.J., Park, C.J., Park, J.E., Cho, Y.W., Veenstra, T.D., Thorner, J., and Lee, K.S. (2004). Coupling morphogenesis to mitotic entry. Proc. Natl. Acad. Sci. USA 101, 4124–4129. Smolka, M.B., Albuquerque, C.P., Chen, S.H., and Zhou, H. (2007). Proteomewide identification of in vivo targets of DNA damage checkpoint kinases. Proc. Natl. Acad. Sci. USA 104, 10364–10369. Stray, J.E., Crisona, N.J., Belotserkovskii, B.P., Lindsley, J.E., and Cozzarelli, N.R. (2005). The Saccharomyces cerevisiae Smc2/4 condensin compacts DNA into (+) chiral structures without net supercoiling. J. Biol. Chem. 280, 34723–34734. Sullivan, M., Higuchi, T., Katis, V.L., and Uhlmann, F. (2004). Cdc14 phosphatase induces rDNA condensation and resolves cohesin-independent cohesion during budding yeast anaphase. Cell 117, 471–482. Sutani, T., Yuasa, T., Tomonaga, T., Dohmae, N., Takio, K., and Yanagida, M. (1999). Fission yeast condensin complex: essential roles of non-SMC subunits for condensation and Cdc2 phosphorylation of Cut3/SMC4. Genes Dev. 13, 2271–2283. Swedlow, J.R., and Hirano, T. (2003). The making of the mitotic chromosome: modern insights into classical questions. Mol. Cell 11, 557–569. Takemoto, A., Murayama, A., Katano, M., Urano, T., Furukawa, K., Yokoyama, S., Yanagisawa, J., Hanaoka, F., and Kimura, K. (2007). Analysis of the role of Aurora B on the chromosomal targeting of condensin I. Nucleic Acids Res. 35, 2403–2412. Torres-Rosell, J., Machin, F., Jarmuz, A., and Aragon, L. (2004). Nucleolar segregation lags behind the rest of the genome and requires Cdc14p activation by the FEAR network. Cell Cycle 3, 496–502. Ubersax, J.A., Woodbury, E.L., Quang, P.N., Paraz, M., Blethrow, J.D., Shah, K., Shokat, K.M., and Morgan, D.O. (2003). Targets of the cyclin-dependent kinase Cdk1. Nature 425, 859–864. Uemura, T., Ohkura, H., Adachi, Y., Morino, K., Shiozaki, K., and Yanagida, M. (1987). DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe. Cell 50, 917–925. Wang, B.-D., Yong-Gonzalez, V., and Strunnikov, A. (2004). Cdc14p/FEAR pathway controls segregation of nucleolus in S. cerevisiae by facilitating condensin targeting to rDNA chromatin in anaphase. Cell Cycle 3, 960–967. Wang, J.C. (1996). DNA topoisomerases. Annu. Rev. Biochem. 65, 635–692. Yeong, F.M., Lim, H.H., Padmashree, C.G., and Surana, U. (2000). Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20. Mol. Cell 5, 501–511.