Supplementary Figure 1 Mutational analysis of the SA2-Scc1 interaction in vitro and in human cells. (a) Autoradiograph (top) and Coomassie stained gel (bottom) of 35 S-labeled Myc-SA2 proteins (input) and the same proteins bound to GST or GST-Scc1 beads. WT, wild type. (b) Quantification of the in vitro binding assays in a. The binding intensities were normalized to the amount of input for each SA2 protein. Error bars, s.d. (n = 3, independent experiments). The Y331A, Y479A, K1009E L1010A mutants were tested twice. Only the means were shown for these samples. (c) Autoradiograph (top) and Coomassie stained gel (bottom) of 35 S-labeled Scc1-Myc proteins (input) and the same proteins bound to GST or GST-SA2 beads. WT, wild type. (d) Quantification of the in vitro binding assays in c. The binding intensities were normalized to the amount of input for each Scc1 protein. Error bars, s.d. (n = 3, independent experiments). (e) Anti-Myc or anti-gfp immunoblots of lysates and anti-myc IP of HeLa cells cotransfected with plasmids encoding GFP-SA2 and the indicated Myc-Scc1 proteins. WT, wild type.
Supplementary Figure 2 Identification of a Sgo1-binding site on SA2 Scc1. (a,b) Quantification of normalized intensities of the indicated 35 S-labeled SA2 Scc1 proteins bound to beads coupled to phospho-t346 Sgo1 peptides. WT, wild type. Error bars, s.d. (n = 3, independent experiments). (c) Anti-Myc, anti-gfp, and anti-sgo1 blots of lgg and anti-gfp IP from GFP-Sgo1-expressing HeLa cells co-transfected with Scc1-Myc and the indicated Myc-SA2 plasmids. WT, wild type. (d) Anti-Myc, anti-sa2, and anti-tubulin blots of lysates of HeLa cells transfected with the indicated sirnas and plasmids. WT, wild type. (e) Quantification of mitotic indices (defined as MPM2-positive, 4N cells) of cells in d. Error bars, s.d. (n = 7, independent experiments).
Supplementary Figure 3 The conserved FVHRYRD motif of SA2 is not required for cohesin loading in human cells. (a) Cartoon diagram of the structure of human SA2 Scc1, with SA2 colored blue and Scc1 colored pink. The conserved SA2 Scc1 regions implicated in binding the cohesin loader Scc2 Scc4 are colored yellow. The MES molecule bound near the SA2 Scc1 interface is shown in sticks. The chemical structure of MES is shown in the lower right corner. (b) A zoomed-in view of the MESbinding site, with the kicked OMIT map of electron density around MES shown at a contour level of 3σ. (c) DAPI (blue in merge), anti- GFP (red in merge), and anti-tubulin (green in merge) staining of telophase HeLa cells transfected with the indicated GFP-SA2 plasmids and sirnas. WT, wild type. Scale bar, 10 µm. (d) Quantification of the anti-gfp staining intensities of cells in c. Each dot in the graph represents a single cell (Mock, n = 12; Vector, n = 22; WT, n = 48; Y297A, n = 18; R298E, n = 46; D793K, n = 40). The horizontal bars indicate the means.
Supplementary Figure 4 Identification of a Wapl-binding site on SA2 Scc1. (a) Anti-Wapl and anti-tubulin blots of lysates of HeLa cells transfected with the indicated sirnas with or without increasing amounts of the indicated GFP-Wapl plasmids. WT, wild type. The positions of the endogenous and GFP-Wapl are labeled. (b) Quantification of mitotic indices (defined as MPM2-positive, 4N cells) of cells in a. Error bars, range (n = 2, independent experiments). (c) Autoradiograph (top) and Coomassie stained gel (bottom) of 35 S-labeled Myc-SA2 Scc1 proteins (input) and the same proteins bound to beads containing GST or increasing amounts of GST-Wapl-M. WT, wild type. (d) Quantification of the in vitro binding assays in c. The binding intensities were normalized to the amount of each input. Error bars, range (n = 2, independent experiments). The K290E and D326K mutants were only tested once.
Supplementary Figure 5 Expression of Wapl binding deficient SA2 mutants bypasses Sgo1 requirement in cohesion protection and rescues cohesion fatigue. (a) Anti-SA2, anti-myc, and anti-tubulin blots of lysates of HeLa cells transfected with the indicated sirna and plasmids. WT, wild type. (b) Four major types (I-IV) of metaphase spreads of cells in a, stained with DAPI (blue) and the kinetochore marker CREST (red). Selected sister chromatids were magnified and shown in insets. Scale bar, 5 µm. (c) Quantification of the percentage of cells in a with type III and IV chromosome morphologies as in b. Error bars, range (n = 2, independent experiments). The K330E mutant was tested only once. (d) Quantification of the percentages of mitotic HeLa cells (transfected with the indicated Myc-SA2 plasmids, arrested in nocodazole, and released into medium containing MG132 for 2 hrs) that had types III/IV chromosome morphology. WT, wild type. Error bars, s.d. for mock, WT, D326K (n = 3, independent experiments); range for K330E and Y331A (n = 2, independent experiments).
Supplementary Figure 6 Uncropped original images of gels, autoradiographs, and blots presented in the main figures of this study.