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1.
Figure 5

Figure 5. Depletion of Scc3 and Pds5 does not affect cohesin association with chromatin.. From: Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA.

Yeast strains were staged in G1 with α-factor and released into media with nocodazole. Chromosomal spreads were prepared as in Figure 2. At every time point fluorescence of 50 nuclei was determined. Error bars represent standard deviation. FACS analysis of cellular DNA content is shown in Figure S13. The strains were in (A): 1813 (SCC1-Myc18, SCC3-HA6), 1625 (SCC1-Myc18, SCC3-HA6-degron), 1815 (SCC1-Myc18, PDS5-HA6), 1818 (SCC1-Myc18, PDS5-HA6-degron), in (B): 1771 (SCC3-Myc18, PDS5-HA6), 1796 (SCC3-Myc18, PDS5-HA6-degron), 1734 (PDS5-MYC18, SCC3-HA6) and 1744 (PDS5-Myc18, SCC3-HA6-degron) in (C): 1479 (SCC3-HA6), 1864 (SCC3-HA6, Δwpl1), 1677 (PDS5-HA6), 1866 (PDS5-HA6, Δwpl1), 10589 (SCC1-Myc18) and 1906 (SCC1-Myc18, Δwpl1).

Irina Kulemzina, et al. PLoS Genet. 2012 August;8(8):e1002856.
2.
Figure 4

Figure 4. Interaction of Pds5, Scc3 and Wpl1 with cohesin ring.. From: Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA.

Lysates of nocodazole/benomyl arrested yeast cultures were incubated with IgG sepharose to precipitate Scc1-TAP or Smc3-TAP. The presence of different proteins on the IgG beads was analysed by Western blot probed with anti-HA (12CA5), anti-MYC (71D10) and PAP (P1291, Sigma). The strains were in (A): 1771 (SCC3-MYC18, PDS5-HA6), 1829 (SCC3-MYC18, PDS5-HA6-degron, SCC1-TAP), 1958 (SCC3-MYC18, PDS5-HA6, SCC1-TAP); in (B): 1734 (PDS5-MYC18, SCC3-HA6), 1834 (PDS5-MYC18, SCC3-HA6-degron, SCC1-TAP), 1956 (PDS5-MYC18, SCC3-HA6, SCC1-TAP); in (C): 1882 (WPL1-MYC18, PDS5-HA6), 2014 (WPL1-MYC18, PDS5-HA6, SCC1-TAP), 2016 (WPL1-MYC18, PDS5-HA6-degron, SCC1-TAP); in (D): 1880 (WPL1-MYC18, SCC3-HA6), 2012 (WPL1-MYC18, SCC3-HA6, SCC1-TAP), 2018 (WPL1-MYC18, SCC3-HA6-degron, SCC1-TAP); in (E): 1771 (SCC3-MYC18, PDS5-HA6), 2251 (SCC3-MYC18, PDS5-HA6, SMC3-TAP), 2290 (SCC3-MYC18, PDS5-HA6-degron, SMC3-TAP); in (F): 1734 (PDS5-MYC18, SCC3-HA6), 2249 (PDS5-MYC18, SCC3-HA6, SMC3-TAP), 2264 (PDS5-MYC18, SCC3-HA6-degron, SMC3-TAP); in (G): 1882 (WPL1-MYC18, PDS5-HA6), 2253 (WPL1-MYC18, PDS5-HA6, SMC3-TAP), 2265 (WPL1-MYC18, PDS5-HA6-degron, SMC3-TAP); in (H): 1882 (WPL1-MYC18, PDS5-HA6), 2261 (WPL1-MYC18, SCC3-HA6, SMC3-TAP), 2271 (WPL1-MYC18, SCC3-HA6-degron, SMC3-TAP).

Irina Kulemzina, et al. PLoS Genet. 2012 August;8(8):e1002856.
3.
Figure 1

Figure 1. Scc3, Pds5, and Wpl1 form a complex and associate with Scc1 “core” subunit of cohesin.. From: Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA.

(A) Purified recombinant Scc3, Pds5 and Wpl1 proteins were mixed as indicated and separated by ultracentrifugation on a 10–30% glycerol gradient. A total of 44 gradient fractions were collected and analyzed on a Coomassie-stained 6% SDS-PAGE. Only fractions containing the proteins are shown. Positions of molecular size markers are indicated. (B) Recombinant His6-tagged Scc3, Pds5, and Wpl1 were mixed together and incubated with glutathione-agarose beads charged with GST or GST-fused to the N-terminal (aa 1–168), middle (aa 169–337) or C-terminal (aa 338–566) regions of Scc1. Beads eluates were analyzed by Western blotting with Penta-His antibody (QIAGEN) (upper panel). Coomassie-stained GST-beads are shown in the lower panel.

Irina Kulemzina, et al. PLoS Genet. 2012 August;8(8):e1002856.
4.
Figure 7

Figure 7. Depletion of Scc3, Pds5, and Wpl1 does not affect cohesin turnover rate on chromosomes.. From: Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA.

(A) A fluorescence recovery after photobleaching experiment was performed on mitotic cells of 2353 (wild type), 2390 (SCC3-HA6-degron), 2389 (PDS5-HA6-degron), and 2391 (Δwpl1) strains with endogenous SCC1 tagged with GFP. No recovery of the bleached pericentric cohesin was observed during the experiment. (B) Scc3 and Pds5 stably associate with chromatin. A FRAP experiment was performed on mitotic cells of 2281 (SCC3-GFP) and 2417 (PDS5-GFP) strains. Histone H2B-GFP strain (1904) was used as control. No recovery of bleached Scc3-GFP and Pds5-GFP was observed during experiment in contrast with H2B-GFP. The mean and standard deviation were calculated from independent experiments (numbers of observed cells for each strain are indicated on the graphs).

Irina Kulemzina, et al. PLoS Genet. 2012 August;8(8):e1002856.
5.
Figure 6

Figure 6. Genome-wide distribution of cohesin in the strains depleted of Scc3, Pds5, and Wpl1.. From: Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA.

Scc1-Myc18 ChIP was performed with nocodazole arrested 10589 (wild type, SCC1-Myc18), 1625 (SCC3-HA6-degron, SCC1-Myc18), 1818 (PDS5-HA6-degron, SCC1-Myc18), and 1906 (Δwpl1, SCC1-Myc18) strains. Untagged 1021 (wild type) strain was used as a control to determine signal log ratio. A running 500 bp window with a 50 bp step size was used to estimate local Scc1 abundance on chromosomal DNA. (A) Scatter plot between chromosomal Scc1 distributions in wild type vs SCC3-degron, PDS5-degron and Δwpl1 strains. Regions corresponding to WPL1 gene that are absent in Δwpl1 strain are circled. (B) Scc1 distribution in the pericentromeric regions of chromosomes 3 and 5. Position of the core centromere is marked with an arrow.

Irina Kulemzina, et al. PLoS Genet. 2012 August;8(8):e1002856.
6.
Figure 8

Figure 8. Cohesin rings devoid of Scc3 and Pds5 topologically embrace circular DNA.. From: Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA.

Strains 1021 (untagged), 1813 (SCC3-HA6, SCC1-Myc18), 1625 (SCC3-HA6-degron, SCC1-Myc18), 2525 (PDS5-HA6, SCC1-Myc18) and 1818 (PDS5-HA6-degron, SCC1-Myc18) carried the centromeric minichromosomes. (A) Yeast lysates were incubated with BglII restriction enzyme as indicated. Minichromosomes were co-immunoprecipitated with Scc1-Myc18. DNA was prepared by phenol/chloroform extraction and separated on a 1% agarose gel with ethidium bromide. Southern blot probed with a TRP1-specific probe is shown. Nicked (N), linear (L), and closed circular (C) forms of the minichromosome are indicated. (B) Minichromosomes were immunoprecipitated with anti-HA antibody. Minichromosomes from SCC3-HA6 but not SCC3-HA6-degron strains could be co-immunoprecipitated with Scc3 indicating the efficient depletion of Scc3 from the minichromosomes in the SCC3-HA6-degron strain. Since Pds5 association with minichromosomes is very salt-sensitive, they could not be co-immunoprecipitated with Pds5-HA6 in either the wild type or PDS5-HA6-degron strains under our experimental conditions.

Irina Kulemzina, et al. PLoS Genet. 2012 August;8(8):e1002856.
7.
Figure 10

Figure 10. A model of how Scc3 and Pds5 play a role in the establishment of sister chromatid cohesion but are not required to stabilize cohesin rings on the DNA.. From: Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA.

In the normal cell cycle of budding yeast Scc1 subunit is synthesized in the late G1 or early S phase. Scc1 binds to Smc1/Smc3 heterodimer and completes the cohesin ring. Scc3 and Pds5 stably associate with cohesin via Scc1 subunit. Cohesin complexes are loaded on the chromosomes. During DNA replication two newly generated sister chromatids are captured inside a single cohesin ring in a process which remains poorly understood. Scc3 and Pds5 function to ensure that two sister chromatids are captured inside a cohesin ring. In their absence, cohesin complexes are stably loaded on the DNA but fail to embrace both of the sister chromatids resulting in defective sister chromatid cohesion (A). We can speculate that Pds5 and Scc3 could stabilize cohesin rings specifically during the replication fork passage (B) or transiently bind sister chromatids during the establishment of cohesion (C). Alternatively they could mediate a transient interaction between two cohesin rings as proposed by [61] (D).

Irina Kulemzina, et al. PLoS Genet. 2012 August;8(8):e1002856.
8.
Figure 2

Figure 2. Depletion of Scc3 and Pds5 with a “conventional” temperature-sensitive degron.. From: Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA.

(A–C) Strains 2395 (SCC1-HA6), 2452 (SCC1-HA6, degron-MYC18-PDS5), 2455 (SCC1-HA6, degron-MYC18-SCC3) and 2456 (SCC1-HA6, degron-MYC18- PDS5, degron-MYC18-SCC3) were arrested with nocodazole in YEP raffinose at 30°C for 2 hours, resuspended in YEP galactose containing nocodazole and incubated for 45 minutes at 30°C to induce the expression of Ubr1. Cells were shifted to 37°C in YEP galactose containing nocodazole and doxycycline to deplete Pds5 and/or Scc3. (A) Chromosomal spreads were prepared at the indicated time points and stained with DAPI for DNA, anti-HA (mouse, 16B12) and anti-MYC (rabbit, 71D10) antibodies. The secondary antibodies were Alexa Fluor 488 anti-mouse and Alexa Fluor 568 anti-rabbit. Protein fluorescence was quantified using Metamorph software. At every time point fluorescence of 50 nuclei was determined. Error bars represent standard deviation. (B) Western blot of TCA protein extracts probed with anti-HA (16B12), anti-MYC (71D10) and anti-Cdc28 (sc-28550, Santa Cruz). (C) FACS analysis of cellular DNA content. (D–F) Strains were staged in G1 with α-factor in YEP raffinose at 30°C, resuspended in YEP galactose containing α-factor and incubated for 45 minutes at 30°C to induce the expression of Ubr1. Cells were then shifted to 37°C in YEP galactose containing doxycycline and α-factor, incubated for 90 minutes to deplete Pds5 and/or Scc3 and subsequently released in YEP galactose containing nocodazole and doxycycline at 37°C. Chromosomal spreads (D), Western blot (E), and FACS analysis of cellular DNA content (F) are shown.

Irina Kulemzina, et al. PLoS Genet. 2012 August;8(8):e1002856.
9.
Figure 9

Figure 9. Sister chromatid cohesion defect in yeast depleted of Scc3 and Pds5.. From: Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA.

(A) Yeast strains 1417 (wild type), 2190 (Δwpl1), 1621 (SCC3-degron) and 1678 (PDS5-degron) have an array of 200 Tet operators integrated into URA3 locus 35 kb from the centromere on chromosome V and express TetR-GFP. Cells were staged in G1 with α-factor and released into media with nocodazole for 100 minutes. Separation of sister chromatids was scored as one (non-separated) versus two (separated) GFP dots. Dots separation was scored in 300 cells per strain. (B) 1759 (wild type), 1776 (SCC3-degron), 1779 (PDS5-degron) and 1769 (Δwpl1) strains with Myc-tagged endogenous SMC3 gene were released from α-factor arrest into nocodazole containing media. Samples were collected at the indicated time points and processed for Western blot probed with anti-acetyl-Smc3 antibody (see Figure S11). The same blot was re-probed with anti-MYC to detect Smc3 (71D10). Western blots with different amounts of samples are shown to demonstrate both the actual levels of Ac-Smc3 in different strains and equal loading. FACS analysis of cellular DNA content is shown. (C) 1822 (wild type), 2436 (Δwpl1), 1832 (SCC3-degron) and 1833 (PDS5-degron) strains with CENIV GFP dots and Spc42-Tomato were expressing Cdc20 from methionine-repressible promoter. Strains were arrested in G1 with α-factor and methionine was then added for 1 hour to shut down Cdc20 expression. Cells were released from G1 arrest into methionine and nocodazole containing media for 2 hours. Microtubule poisons were washed out and samples taken at indicated times, fixed in methanol and analyzed by fluorescence microscopy.

Irina Kulemzina, et al. PLoS Genet. 2012 August;8(8):e1002856.
10.
Figure 3

Figure 3. Eco1 contains a degron.. From: Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA.

(A) 1222 strain with CDC20 expressed from a methionine-repressible promoter (ECO1-TAP) was staged in G1 with α-factor and then released into three different media. Release into the medium without methionine allows cell cycling (top) while release into full medium with methionine or nocodazole (middle and bottom) results in progression through replication and eventual G2 arrest. Expression of S phase cyclin Clb5 is induced during DNA replication while cyclin Clb2 accumulates in G2 and is destroyed in mitosis. TCA protein extracts were prepared and analyzed by Western blot. Eco1 was detected with peroxidase-anti-peroxidase, Clb5 with sc-6704, Clb2 with sc-9071, loading control with anti-Cdc28 (sc-28550, Santa Cruz). (B) Depletion of Scc3 and Pds5 with an Eco1-derived degron. G1-staged strains 12544 (SCC3-HA6), 1323 (SCC3-HA6-degron), 1677 (PDS5-HA6) and 1675 (SCC3-HA6-degron) were released into full media with nocodazole. Western blot was probed with anti-HA antibody. (C) Scc1 protein level is unchanged in the strains with Eco1-derived degron. Strains 1815 (SCC1-Myc18, PDS5-HA6), 1818 (SCC1-Myc18, PDS5-HA6-degron), 1813 (SCC1-Myc18, SCC3-HA6), 1625 (SCC1-Myc18, SCC3-HA6-degron), 10589 (SCC1-MYC18) and 1906 (SCC1-MYC18, Δwpl1) were staged in G1 with α-factor and released into media with nocodazole. Western blot was probed with anti-HA, anti-Myc and anti-Cdc28 antibodies. The same yeast cultures were used for chromosomal spreads (Figure 5) and for FACS analysis of cellular DNA content (Figure S13A). (D and E) Determination of Scc3 and Pds5 copy number per yeast cell. (D) Coomassie-stained gel with serial dilutions of purified recombinant Scc3-HA6, Pds5-HA6 and BSA (NEB #B9001) as a standard. (E) Protein extracts were prepared from nocodazole-arrested strains 1323 (SCC3-HA6-degron), 1479 (SCC3-HA6), 1675 (PDS5-HA6-degron) and 1677 (PDS5-HA6). Extracts from the indicated number of cells were analyzed by Western blot with anti-HA antibody. Known quantities of recombinant Scc3-HA6 and Pds5-HA6 were used as standards. Bands were quantified with MetaMorph.

Irina Kulemzina, et al. PLoS Genet. 2012 August;8(8):e1002856.

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