1. Volume 33, Issue 6, Pages 763-774 (March 2009)
Building Sister Chromatid Cohesion: Smc3 Acetylation Counteracts an Antiestablishment Activity 
Benjamin D. Rowland, Maurici B. Roig, Tatsuya Nishino, Alexander Kurze, Pelin Uluocak, Ajay Mishra, Frédéric Beckouët, Philippa Underwood, Jean Metson, Richard Imre, Karl Mechtler, Vittorio L. Katis, Kim Nasmyth 
Molecular Cell 
Volume 33, Issue 6, Pages (March 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1
eco1-1 Suppressor Mutations Identify Protein Domains that Regulate Establishment of Sister Chromatid Cohesion
(A) Thirteen dominant mutations map to ECO1 and 206 recessive or partially recessive mutations belong to four complementation groups, smc3, scc3, pds5, and rad61.
(B) Amino acid changes identified by DNA sequencing. Several mutations were identified more than once. Frameshift mutations, resulting in premature stop-codons, are depicted by an “x”.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

3. Figure 2
Suppressor Mutations Permit Growth of eco1Δ Cells and Cohesion between Circular Sister Minichromosomes in ts eco1 Mutants
(A) Tetrad dissection of heterozygous ECO1/eco1Δ strains in either a wild-type background (K15889) or in a background heterozygous for suppressor mutations smc3 K113T (K15890), scc3 E202K (K15891), pds5 S81R (K15892), or rad61Δ (K15893). The encircled spore clones bear the deletion marker for eco1Δ. Three representative tetrads are shown from a minimum of 40 dissections of each strain.
(B) Extracts from wild-type (K16102), eco1 G211H (K16107), and eco1 G211H pds5 S81R (16110) strains harboring a 2.3 kb minichromosome and which had undergone DNA replication at 37°C were fractionated by sucrose gradient centrifugation followed by gel electrophoresis. Minichromosome DNA was detected by southern blotting. Dimers are marked with a red box.
(C) The percentage of minichromosome DNA present in dimer fractions in wild-type (K16102), ECO1 smc3 K113T (K16103), ECO1 scc3 E202K (K16104), ECO1 pds5 S81R (K16105), ECO1 rad61Δ (K16106), eco1 G211H (K16107), eco1 G211H smc3 K113T (K16108), eco1 G211H scc3 E202K (K16109), eco1 G211H pds5 S81R (16110), and eco1 G211H rad61Δ (K16111).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. Figure 3
Suppressor Mutations Cause Cohesion Defects in a Wild-Type Background
(A) Percentage of cells with single or double (split) Tet operator/repressor GFP dots at the URA3 locus (ura3::3xURA3 tetO112; tetR-GFP) in wild-type (K15747) or mutant smc3 G110W (K15745), smc3 K113T (K15746), scc3 E202K (K15741), scc3 R1043L (K15742), pds5 P89S (K15743), pds5 E602K (K15744), rad61Δ (K15748), and rad61Δ eco1Δ (K15749) cells arrested in metaphase by depleting Cdc20. Cells growing in minimal medium at 30°C lacking methionine were transferred to YEPD plus methionine, which induced uniform metaphase arrest after 3 hours.
(B) Chromatin immunoprecipitation (ChIP) followed by quantitative PCR on asynchronously proliferating cells. PK ChIPs were performed on a nontagged strain (K699) and on strains expressing Scc1-PK9 in a wild-type (K14601) or rad61Δ (K15882) background. Shown is the percentage (of input DNA) within ChIPs measured by qPCR at core centromere, pericentromere, and arm sequences. Data are depicted as the average and standard deviations of four independent experiments.
(C) Myc18-tagged Rad61 associated with three chromosomal loci (as measured by ChIP and qPCR) in wild-type (K15721) and temperature-sensitive scc1-73 cells (K15677) as MATa cells are released from α factor-induced G1 arrest in fresh medium at 37°C. Cell-cycle progression was assessed by FACS analysis (right). An untagged strain (K699) yielded no significant signal.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

5. Figure 4 Rad61 Binds to Both Scc3 and Pds5
(A) Reconstitution of the Scc3/Rad61/Pds5 complex. Rad61, Scc3, or Pds5 were incubated either alone or in combination at 15 μM of each protein in 0.1 M NaCl, separated by gel filtration, and analyzed by SDS-PAGE. The respective fractions from the gel filtrations are depicted above each lane. Rad61 efficiently associates with Scc3 and with Pds5. When mixed together, all three proteins form a stable trimeric complex.
(B) Scc3 and Pds5 do not stably interact in the absence of Rad61 at 0.1 M NaCl. The experiment was performed as in (A).
(C) Rad61 binds to Scc3 and to Pds5 in vivo. Immunoprecipitations and western blots on extracts of an untagged strain (K699) or strains with the following C-terminal tags: Pds5-HA6 (K16275), Scc3-HA6 (K16274), Rad61-Myc18 (K15721), Pds5-HA6/Rad61-Myc18 (K15726), and Scc3-HA6/Rad61-Myc18 (K15935) using either HA or Myc antibodies. Swi6 serves as a loading control.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

6. Figure 5
Suppressor Mutations in SMC3 Cluster in a Patch on its NBD, which Is Acetylated by Eco1
(A) Predicted structure of the Smc3 head domain complexed with the known structures for the Smc1 head and the C terminus of Scc1 (Haering et al., 2004). Pyrococcus furiosus Smc (Lammens et al., 2004) was used as a template for modeling of the Smc3 head. ATP is visualized in yellow. The smc3 suppressor mutations and the conserved lysines 112 and 113 are indicated in orange.
(B) Homology alignment of the domain of Smc3 that contains the suppressor mutations. The mutated residues are indicated in red, as are lysines 112 and 113.
(C) The head domain of Smc3 is acetylated in vitro by Eco1. Purified fragments of Scc1 and Smc3 proteins were incubated with Eco1 protein in the presence of 14C-labeled Acetyl-CoA, and analyzed by SDS-PAGE followed by autoradiography (left) or Coomassie staining (right).
(D) Lysines K112 and K113 are acetylated by Eco1 in vitro. Experiment performed as in (C), but with unlabeled Acetyl-CoA. Proteins were gel-purified and analyzed by mass spectrometry.
(E) Smc3 is acetylated in vivo at lysines 112 and 113 in an Eco1-dependent manner. Full-length Smc3 was purified from asynchronously proliferating smc3Δ SMC3-HA3 cells (K14989: ECO1 RAD61, K15793: ECO1 rad61Δ, and K15794: eco1Δ rad61Δ) and analyzed by tandem mass spectrometry (MS/MS).
(F) The experiment performed as in (E), but analyzed by western blot using an acetyl-lysine-specific antibody.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

7. Figure 6
Lethality Due to Mutating Smc3's Lysines 112 and 113 to Arginines Is Rescued by Suppressor Mutations in SCC3, PDS5, or RAD61
(A) Tetrad dissections were performed on heterozygous SMC3/smc3Δ strains with smc3 K112R K113R integrated at the leu2 locus, either in a wild-type background (K15894) or in a background heterozygous for the respective suppressor mutants scc3 E202K (K15896), pds5 S81R (K15897), or rad61Δ (K15895). Spores that have the integrated copy of SMC3 as their sole copy of SMC3 are encircled. These spores also carry the depicted suppressor mutations in SCC3, PDS5, or the deletion of RAD61. Three representative tetrads are shown from a minimum of 40 dissections of each strain.
(B) Smc3 acetylation in G2/M requires continued Eco1 activity. Smc3-HA6 was immunoprecipitated from wild-type (K12639) or eco1-1 G211D (K7672) cells that were arrested in nocodazole for 100 min at 23°C and then shifted to 37°C for 120 min. Western blots were performed using either HA-specific or acetyl-lysine-specific antibodies.
(C) Smc3 (K112R; K113R) has equal turnover rate compared to wild-type Smc3. Fluorescence microscopy of yeast cells expressing ectopic Smc3-GFP (K16254) or Smc3 (K112R; K113R)-GFP (K16245). H2B-GFP (K16112) serves as a control. Red circles depict bleached areas. Data points and error bars are based on the average and standard deviations of eight independent FRAP experiments. Intensities were normalized to the fluorescence levels before photobleaching. Curves were fitted to data points starting at the time point immediately after the bleach.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

8. Figure 7 Models for Establishment and Antiestablishment
(A) Eco1 counteracts antiestablishment activity of Scc3/Rad61/Pds5 proteins. Either Eco1 counteracts an antiestablishment activity of Scc3/Rad61/Pds5 that hinders the establishment of cohesion. Eco1 allows establishment of cohesion by counteracting this inhibition (1). Or Eco1 counteracts an activity of Scc3/Rad61/Pds5 proteins that destabilizes cohesin's association with DNA after it has entrapped sister chromatids (2).
(B) Antiestablishment might be achieved through direct binding of the Scc3/Rad61/Pds5 complex to the K112/K113 patch in Smc3. Eco1 counteracts this binding by acetylation of K112 and K113, which results in the coentrapment of sister DNAs. Mutations in either the K112/K113 patch or in the Scc3/Rad61/Pds5 complex have the same effect and allow coentrapment of sister DNAs in the absence of Eco1.
(C) Antiestablishment counteracts the coentrapment of sister DNAs by either of two mechanisms. (Ca) Antiestablishment prevents de novo coentrapment of sister DNAs, or (Cb) antiestablishment prevents conversion from solitary entrapment of a single DNA to coentrapment of both sister DNAs.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions