1. Volume 34, Issue 4, Pages 416-426 (May 2009)
Polo Kinase Regulates Mitotic Chromosome Condensation by Hyperactivation of Condensin DNA Supercoiling Activity 
Julie St-Pierre, Mélanie Douziech, Franck Bazile, Mirela Pascariu, Éric Bonneil, Véronique Sauvé, Hery Ratsima, Damien D'Amours 
Molecular Cell 
Volume 34, Issue 4, Pages (May 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 Phosphorylation of Condensin in Anaphase
(A–C) D506 (BRN1-3HA cdc15-2), D517 (YCG1-3HA cdc15-2), and D491 (YCS4-13MYC cdc15-2) cells were arrested in G1 in YEPD medium containing α factor (5 μg/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, BRN1-3HA cdc15-2; D930, YCG1-3FLAG cdc15-2; D491, YCS4-13MYC cdc15-2) and processed for western analysis or dephosphorylation by λ phosphatase.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

3. Figure 2 Phosphorylation of Condensin Depends on Polo/Cdc5 Kinase
Cells were arrested in G1 in YEPD medium with α 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).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. Figure 3
Condensin Phosphorylation Is Partially Dependent on Aurora B/Ipl1 Kinase
Cells were arrested in G1 in YEPD medium with α 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).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

5. Figure 4 Cdc5 Is a Condensin Kinase
(A) D506 (BRN1-3HA cdc15-2), D517 (YCG1-3HA cdc15-2), and D491 (YCS4-13MYC 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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

6. 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 λ phosphatase.
(H–J) Cells were arrested in G1 in YEPD medium with α 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 (brn HA cdc15-2).
(I) D491 (YCS4-13MYC cdc15-2) and D1165 (ycs MYC cdc15-2).
(J) D517 (YCG1-3HA cdc15-2), D1135 (ycg HA cdc15-2), and D1139 (ycg HA 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 (ycg HA cdc15-2), D506 (BRN1-3HA cdc15-2) and D1231 (brn HA cdc15-2), and D491 (YCS4-13MYC cdc15-2) and D1165 (ycs MYC 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 32P-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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

7. 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 (brn HA cdc15-2), D491 (YCS4-13MYC cdc15-2), D1165 (ycs MYC cdc15-2), D517 (YCG1-3HA cdc15-2), D1135 (ycg HA cdc15-2), D1138 (ycg HA cdc15-2), D1139 (ycg HA cdc15-2), and D1088 (ycg HA 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 (brn HA ycg HA ycs MYC 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).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

8. 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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions