Volume 127, Issue 3, Pages 523-537 (November 2006) Evidence that Loading of Cohesin Onto Chromosomes Involves Opening of Its SMC Hinge  Stephan Gruber, Prakash Arumugam, Yuki Katou, Daria Kuglitsch, Wolfgang Helmhart, Katsuhiko Shirahige, Kim Nasmyth  Cell  Volume 127, Issue 3, Pages 523-537 (November 2006) DOI: 10.1016/j.cell.2006.08.048 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Connection of Scc1 with Either SMC Head Permits Cohesion (A) Models for cohesin ring opening. (B) Scc1-Smc1 fusion protein expression. Cells of strains K699 (MATa) and K11680 (MATa PSCC1SCC1-TEV3-SMC1-myc18 Δscc1 Δsmc1) were grown in exponential phase (cyc) and arrested in metaphase using nocodazole (M) or in G1 phase by addition of α factor peptide (G1). Cell extracts were prepared and analyzed by blotting for myc and Swi6 protein. For detailed genotypes, see Table S1. (C) SCC1-SMC1 is functional. Genotypes of spores (germinated on YPD plates at 30°C) from diploid yeast strains K12314 (MATa/α SCC1/Δscc1 SMC1/Δsmc1) and K13630 (MATa/α SCC1/Δscc1 SMC1/Δsmc1 leu2/leu2::PSCC1SCC1-TEV3-SMC1::LEU2) are shown. (D) SCC1-SMC1 supports cell growth. Five-fold serial dilutions of strains K699 (wt) and K12385 (MATa Δscc1 Δsmc1 PSCC1SCC1-TEV3-SMC1) (fu) were plated onto YPD and grown at 30°C. (E) SCC1-SMC1 cells are resistant to CScc1. Strains K11969 (MATa YCp-PGAL1CScc1-HA3) (wt) and K11970 (MATa Δscc1 Δsmc1 PSCC1SCC1-TEV3-SMC1-myc18 YCp-PGAL1CScc1-HA3) (fu) were plated on −TRP plates containing either 2% glucose or 2% raffinose plus galactose and grown at 25°C. Expression of CScc1 (aa 268–566) was confirmed by detecting HA epitopes in blots from extracts of cells shifted for 3 hr from raffinose to glucose (−CScc1) or galactose (+CScc1). The fusion strain grows significantly more slowly than wild-type on −Trp medium, possibly because SCC1-SMC1-myc18 cells more frequently lose the centromeric plasmid. (F) SMC3-SCC1 is functional. Genotypes of spores from diploid yeast strains K12542 (MATa/α SCC1/Δscc1 SMC3/Δsmc3) and K13638 (MATa/α SCC1/Δscc1 SMC3/Δsmc3 ura3/ura3::PSCC1SMC3-TEV3-SCC1::URA3) after dissection on YPD at 30°C. (G) SMC3-SCC1 supports cell growth. Serial dilutions of strains K699 (wt) and K12988 (MATα Δscc1 Δsmc3 PSCC1SMC3-TEV3-SCC1) (fu) plated onto YPD and grown at 30°C are shown. Cell 2006 127, 523-537DOI: (10.1016/j.cell.2006.08.048) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Linking Scc1 to Both SMC Heads Permits Sister-Chromatid Cohesion (A) Linking Scc1's C terminus with Smc1's head by rapamycin-dependent dimerization permits cohesin function. Strains K14708 (MATα Δfpr1 TOR1-1) (wt) and K14195 (MATa Δfpr1 TOR1-1 SCC1-Frb SMC1-FKBP12) (Fk+Fr) were plated on YPD containing 0 or 100 nM rapamycin. Plates were incubated at 30°C. (B) Linking Scc1's C terminus with Smc1's head suppresses mutations in Scc1's C terminus. Strains K14195 (wt), K14196 (MATa Δfpr1 TOR1-1 scc1(S525N)-Frb SMC1-FKBP12) (S525N), and K14197 (MATa Δfpr1 TOR1-1 scc1(Q544K)-Frb SMC1-FKBP12) (Q544K) were grown in the presence of 100 nM rapamycin and plated onto YPD containing 0 or 100 nM rapamycin. (C and D) Linking Scc1 to both SMC heads permits sister-chromatid cohesion. Cells of strains K30016 (MATa Δfpr1 TOR1-1 Δscc1 Δsmc3 PSCC1SMC3-TEV3-SCC1-Frb SMC1-FKBP12) and K14668 (MATa Δfpr1 TOR1-1 Δsmc3 SMC3Frb SMC1FKBP12) were synchronized by centrifugal elutriation and incubation with α factor for 3 hr at 25°C. Cells were released from the arrest and incubated for 3 hr in nocodazole with or without rapamycin. Cells were fixed and subjected to fluorescence in situ hybridization (FISH) using a Cy3-labeled DNA probe covering a 16.6 kb region around the centromere on chromosome IV. (E) Linking Scc1 to both SMC heads does not block cohesin's DNA association. Cells of strain K14829 (MATa Δfpr1 TOR1-1 Δscc1 Δsmc3 PSCC1-SMC3-TEV3-SCC1-Frb SMC1-FKBP12 SCC3-myc18) were synchronized by centrifugal elutriation and arrested with α factor for 3.5 hr at 25°C. Cells were released by washing with YPD medium and incubated in the presence or absence of rapamycin. Replication timing was monitored by flow cytometry. The levels of cohesin associated with DNA were determined by chromatin immunoprecipitation (ChIP) and quantitative PCR using primer pairs specific to three different regions of chromosome VI (see below). Cell 2006 127, 523-537DOI: (10.1016/j.cell.2006.08.048) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 MP1 and p14 Interact Tightly Enough to Resist the Mitotic Spindle (A) Yeast strains K12822 (MATα PMET3CDC20 YEp Δscc1 HA6-SCC1(TEV220)), K12823 (MATα PMET3CDC20 YEp-PGAL1TEV Δscc1 HA6-SCC1(TEV220)), K12824 (MATa PMET3CDC20 YEp Δscc1 HA6-SCC1MP1-(TEV220)-p14), and K12825 (MATa PMET3CDC20 YEp-PGAL1TEV Δscc1 HA6-SCC1MP1-(TEV220)-p14) were grown to log phase in synthetic medium lacking methionine (cyc) and arrested for 2 hr in YEP medium supplemented with 2 mM methionine and 2% raffinose (0 min). Expression of TEV protease was induced by addition of 2% galactose (0–150 min). Cells were transferred back to synthetic medium lacking methionine at 150 min (+Cdc20). Mono- and binucleate cells were counted after staining with DAPI. Protein extracts were prepared and probed by immunoblotting for HA epitopes. (B) Dilution of cells of strains K13653 (MATα Δscc1 HA6-SCC1(TEV220) YEp-PGAL1TEV) and K13654 (MATa Δscc1 HA6-SCC1MP1-(TEV220)-p14 YEp-PGAL1TEV) were plated on synthetic medium lacking tryptophan with glucose or raffinose and galactose. Plates were incubated at 30°C. Cell 2006 127, 523-537DOI: (10.1016/j.cell.2006.08.048) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Hinge-Substituted SMC Heterodimers Form Rings with Scc1 and Hydrolyze ATP with Wild-Type Kinetics but Cannot Associate with Chromosomes (A) Ribbon diagrams of the structures of a SMC hinge homodimer from T. maritima (top) and the MP1-p14 complex from mouse (bottom). Diagrams were produced by Cn3D using the PDB ID codes 1GXL and 1VET, respectively. (B) Hinge-substituted SMC heterodimers form tripartite rings with Scc1. Yeast strains K14084 (MATa/α myc9-SCC1TEV268-myc9/ -PGAL1SCC1/SCC1), K14081 (MATa/α myc9-SCC1TEV268-myc9/(SMC1-myc9+SMC3-HA3) PGAL1SCC1/SCC1), and K14082 (MATa/α myc9-SCC1TEV268-myc9/(smc1p14-myc9+smc3MP1-HA3) PGAL1SCC1/SCC1) were grown to log phase in YPD medium. Cleared extracts (IN) were immunoprecipitated with anti-HA antibodies, and supernatants were removed (FT). The beads were washed (IP) and incubated with or without TEV protease for 45 min at 16°C. Supernatant fractions were collected (SN) and proteins eluted from the beads using SDS (B). Samples were analyzed by immunoblotting using α-HA and α-myc antibodies. (C) Hinge-substituted SMC heterodimers hydrolyze ATP. (Left) Purified SMC heterodimers (5 μg per lane) were separated on a 6% SDS polyacrylamide gel and stained with Coomassie brilliant blue R-250 (1, Smc1 + Smc3; 2, Smc3MP1; 3, Smc1p14). (Right) ATP hydrolysis assays were performed using the same SMC preparations at 0.5 μM and 250 μM ATP in the absence (black curve) or presence (red curve) of 6 μM C-terminal Scc1 fragment. γ32P-labeled ATP and 32Pi were resolved by thin-layer chromatography, and ratios of Pi to Pi + ATP were plotted for each time point. Estimated CScc1-dependent hydrolysis rates are given as molecules of ATP hydrolyzed per minute per SMC heterodimer at 250 μM ATP. The variable levels of activity in the absence of CScc1 presumably stem from different expression and purification efficiencies and represent background activity from impurities. (D) ChIP of wild-type and hinge-substituted SMC proteins. Yeast strains K11990 (MATa Scc1-Pk6), K14022 (MATa Scc1-Pk6 (SMC1-myc9+SMC3-HA3)), and K14024 (MATa Scc1-Pk6 (smc1p14-myc9+smc3MP1-HA3)) were grown in log phase and fixed with 3% formaldehyde. Samples were processed for ChIP analysis using α-myc antibodies. Efficiency of chromatin immunoprecipitation (ratio of input versus IP) was measured by real-time PCR using four primer pairs (for positions on chromosome VI, see below). Error bars indicate standard deviations from the arithmetic mean calculated from two independently processed samples. (E) Distribution of wild-type and hinge-substituted SMCs around the chromosome VI centromere. Yeast strains K13581 (MATa SMC1::(SMC1+SMC3-HA3)) and K13585 (MATa SMC1::(smc1p14+smc3MP1-HA3)) were arrested in mitosis using benomyl. Cells were then processed for ChIP-on-chip analysis using α-HA antibodies. The blue shading represents the binding ratio of loci that show significant enrichment in the immunoprecipitated fraction. The yellow line indicates the average signal ratio of loci that are not enriched in the immunoprecipitated fraction. The scale of the vertical axis is log2. The horizontal axis shows kilobase units (kb). Original ChIP-on-chip data can be accessed from the GEO database under the accession number GSE4827. Cell 2006 127, 523-537DOI: (10.1016/j.cell.2006.08.048) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Linking SMC Hinge Domains by the FKBP12-Frb-Rapamycin Complex Is Lethal (A) Ribbon diagrams of the structures of a FKBP12-rapamycin-Frb complex and a bacterial SMC hinge-domain dimer. Dotted lines represent 10 aa linker peptides that insert FKBP12 into Smc1's hinge domain and Frb into Smc3's hinge domain. (B) Rapamycin-dependent lethality of hinge-inserted SMCs. Yeast strains K14708 (MATα Δfpr1 TOR1-1) (wt), K14645 (MATα Δfpr1 TOR1-1 SMC1FKBP12) (Fk), K14644 (MATa Δfpr1 TOR1-1 Δsmc3 SMC3Frb) (Fr), and K14643 (MATα Δfpr1 TOR1-1 Δsmc3 SMC3Frb SMC1FKBP12) (Fk+Fr) were grown for 2 days on YPD plates in the absence or presence of 100 nM rapamycin. (C) Fpr1 rescues rapamycin-dependent lethality. Yeast strains K11607 (MATa TOR1-1), K14708, K14642 (MATα TOR1-1 Δsmc3 SMC3Frb SMC1FKBP12), and K14643 were grown for 2 days on YPD plates in the absence or presence of 100 nM rapamycin. Cell 2006 127, 523-537DOI: (10.1016/j.cell.2006.08.048) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 SMC Hinge Interconnection Hinders Establishment of Cohesion and Cohesin's Association with Chromosomes (A) Hinge connection hinders establishment, but not maintenance, of cohesion. Cells of strain K14690 (MATa Δfpr1 TOR1-1 Δsmc3 SMC3Frb SMC1FKBP12 PMET3-CDC20 112x tetOs TetR-GFP) were grown in medium lacking methionine and arrested in G1 phase with α factor. Cells were released from the arrest (0 min) into medium supplemented with 2 mM methionine. Replication timing was monitored by flow cytometry (Figure S4B). Rapamycin was added at time point 0 (left graph) or 80 min later (right graph). Cells were stained with DAPI, and sister-chromatid cohesion was monitored by detecting fused or split GFP dots. (B) Hinge connection hinders association of cohesin with chromosomes. Cells of strain K14697 (MATa Δfpr1 TOR1-1 Δsmc3 SMC3Frb SMC1FKBP12 SCC1-Pk9) were grown in YPD medium and arrested in G1 phase with α factor. Cells were released and incubated in YPD medium in the absence or presence of 100 nM rapamycin. Samples were taken every 5 min, and cellular DNA contents were measured by flow cytometry (top panel). Sample aliquots were subjected to chromatin immunoprecipitation using α-Pk-tagged antibodies. Input and ChIP DNA samples were analyzed by real-time PCR using four different primer pairs. Efficiency of pull-down (% chromatin IP) is plotted for each time point. Positions of the primer pairs are indicated in the bottom panel, which shows Scc1 distribution on the central part of chromosome VI in nocodazole-arrested wild-type cells. Cell 2006 127, 523-537DOI: (10.1016/j.cell.2006.08.048) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 A Model for the Transport of DNA through SMC Hinges (A) Crystal structure analysis of the bacterial SMC hinge dimer from T. maritima revealed a closed (left; PDB ID code 1GXK) and a half-open conformation (right; PDB ID code 1GXJ) (Haering et al., 2002). Arrows indicate the directions of the amphipathic helices. (B) A model for transporting DNA into a cohesin ring. Folding of the Smc1 and Smc3 coiled coils might enable interactions of SMC hinge and head domains from the same cohesin ring, possibly creating a DNA binding surface formed by sequences of both hinge and head domains. Upon contact with DNA, binding to or hydrolysis of ATP by the ABC ATPase generates a conformational change in the SMC heads that is transmitted to hinge domains via the coiled coils, the DNA double helix, and/or direct interactions between head and hinge domains, causing disruption of one of the two hinge-hinge contact sites. Entry of DNA into the central channel of the half-open hinge might drive transient detachment of the other SMC hinge-domain interface. Reclosing of the SMC hinge after exit of the DNA double helix would finally trap DNA inside cohesin's ring. Cell 2006 127, 523-537DOI: (10.1016/j.cell.2006.08.048) Copyright © 2006 Elsevier Inc. Terms and Conditions