Volume 63, Issue 6, Pages 1044-1054 (September 2016) A Reversible Association between Smc Coiled Coils Is Regulated by Lysine Acetylation and Is Required for Cohesin Association with the DNA  Irina Kulemzina, Keven Ang, Xiaodan Zhao, Jun-Thing Teh, Vikash Verma, Sasikala Suranthran, Alap P. Chavda, Roland G. Huber, Birgit Eisenhaber, Frank Eisenhaber, Jie Yan, Dmitri Ivanov  Molecular Cell  Volume 63, Issue 6, Pages 1044-1054 (September 2016) DOI: 10.1016/j.molcel.2016.08.008 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 63, 1044-1054DOI: (10.1016/j.molcel.2016.08.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Lysine-to-Arginine Substitutions in the Smc Coiled Coils Result in PSCS (A) Schematic of Smc1 and Smc3 domain composition. The coiled coil domain is divided into high-probability coiled coil (C) and gap (G) segments. Lysines mutated in the head-proximal and hinge-proximal coiled coil mutants are indicated in blue and yellow boxes, respectively. (B–E) The percentage of cells displaying two GFP dots in G1 versus metaphase arrest is presented. The regions of the coiled coil in which all lysines are mutated to arginines are highlighted in gray (B and C) or in blue (D and E). See also Figures S1 and S4 and Tables S1 and S2. Molecular Cell 2016 63, 1044-1054DOI: (10.1016/j.molcel.2016.08.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Smc1 and Smc3 Coiled Coil Mutants Heterodimerize but Are Defective in Scc1 Recruitment and Association with the DNA Cohesin subunits were immunoprecipitated from the lysates of nocodazole-arrested yeast and analyzed by western blot as indicated. Schemes below the figures depict the different protein complexes, which were present in the lysates. Protein complexes that were detected in the IP are boxed in green. The strain numbers are indicated in brackets. (A) Smc1all R heterodimerizes with Smc3all R. (B) Smc1all R/Smc3all R dimers do not recruit Scc1. (C) Smc1cc R/Smc3cc R dimers do not load on the chromosomes. Chromosomal spreads are shown. (D) Smc1cc R/Smc3cc R dimers recruit Scc1. (E and F) Strains expressing photo-crosslinkable Smc3 K318∗BPA or E209∗BPA were UV-irradiated, and Smc1-Myc was detected by western blot. Smc1all R/Smc3all R dimers can be crosslinked at Smc3 K318 (E) but not at Smc3 E209 (F). (G) A model of the Smc1/Smc3 head domain heterodimer showing the position of Smc3 E209. Smc3 head homodimer (PDB: 4UX3) was aligned with the Smc1 head homodimer (PDB: 1W1W). E209 is oriented toward the Smc1 coiled coil when ATP is sandwiched between the head domains. (H) The binding interface of Scc1 with Smc3 is not affected in the smc1core cc R/smc3core cc R mutant. Strains expressing photo-crosslinkable Smc3 A181∗BPA were UV-irradiated, and Scc1-V5 was detected by western blot. Smc3L1029R A181∗BPA, which is defective for Scc1 interaction, is used as a negative control. (I) Crosslinking of the Smc coiled coils is independent of Scc1. Strains expressing photo-crosslinkable Smc3 K318∗BPA or E209∗BPA were arrested with α-factor in G1 (no Scc1) and UV-irradiated. Smc1-Myc was detected by western blot. Fluorescence-activated cell sorting profiles of the cells are shown. See also Figures S1 and S2. Molecular Cell 2016 63, 1044-1054DOI: (10.1016/j.molcel.2016.08.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 SFM Images of Wild-Type and Mutant Cohesins (A) Coomassie-stained gels of cohesin subcomplexes used for the SFM imaging. (B–E) SFM images in air of Smc3 monomers (B), Smc1/Smc3 dimers (C), Smc1/Smc3/Scc1 trimers (D), and Smc1gaps R/Smc3cc R (E). (F) The dimensions of the cohesin subcomplexes based on the SFM data. The error bars are the SD values. The data sets are fitted to a normal distribution. (G) Tilted 3D images. (H) Relative molecular volumes of the Smc3 monomer (79.5 ± 16.1 nm3), Smc1/Smc3 dimer (151.7 ± 36.8 nm3), and Smc1/Smc3/Scc1 trimer (207.3 ± 33.1 nm3) were estimated on the basis of the SFM images and are proportional to their respective molecular weights. The error bars are the SD values. See also Figure S3. Molecular Cell 2016 63, 1044-1054DOI: (10.1016/j.molcel.2016.08.008) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Deacetylation of the Smc1/Smc3 Dimers Promotes Dissociation of Their Arms (A) Recombinant protein complexes were treated with CobB in the absence or presence of NAD+ and loaded on a glycerol gradient. Individual fractions were separated by SDS-PAGE followed by Coomassie staining. Scc1 was detected with the TAP Tag antibody recognizing the C terminus of the TAP construct after TEV cleavage (CAB1001, Thermo). The two Scc1 bands likely represent differentially modified species. The S values of the peak fractions and standards are indicated. (B and C) Peak fractions from (A) were imaged by SFM in air. See also Figure S3. Molecular Cell 2016 63, 1044-1054DOI: (10.1016/j.molcel.2016.08.008) Copyright © 2016 Elsevier Inc. Terms and Conditions