1. Volume 24, Issue 11, Pages 1991-1999 (November 2016)
Impairing Cohesin Smc1/3 Head Engagement Compensates for the Lack of Eco1 Function 
Roland G. Huber, Irina Kulemzina, Keven Ang, Alap P. Chavda, Sasikala Suranthran, Jun-Thing Teh, Dimitar Kenanov, Gaowen Liu, Giulia Rancati, Radoslaw Szmyd, Philipp Kaldis, Peter J. Bond, Dmitri Ivanov 
Structure 
Volume 24, Issue 11, Pages (November 2016)
DOI: /j.str
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2. Structure 2016 24, 1991-1999DOI: (10.1016/j.str.2016.09.001)
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3. Figure 1
The smc3(T1185M) and smc1(S1189A) Mutations Phenotypically Suppress Δeco1
(A) Dynamics of chromosome-bound cohesin. Contact of Smc3 lysines 112 and 113 with the DNA stimulates the ATPase activity of Smc heads and leads to Wpl1-dependent opening of the Smc1/Scc1 interface and release of DNA. Eco1 acetylates Smc3 lysines 112 and 113, inhibiting the releasing activity. Alternatively, the release of DNA from the cohesin ring can be impeded by the point mutations in the Smc heads (indicated by the yellow star), which affect the Smc head engagement.
(B) The conservation of the Smc3-T1185, Smc1-S1189, and H loop among Smc3 and Smc1 orthologs. See also Figure S1.
(C) Effects of smc3(T1185M) and smc1(S1189A) on benomyl sensitivity and temperature sensitivity of Δeco1 Δwpl1. Strain numbers are indicated in parentheses. See also Figure S2A.
(D) smc3(T1185M) and smc1(S1189A) partially rescue the cohesion defect of Δeco1 Δwpl1. Sister chromatid separation was analyzed in strains arrested in G1 (α factor) or metaphase (Cdc20 depletion) and the percentage of cells with two GFP dots was determined. The position of an array of 200 Tet operators is indicated by the green star. WT, wild-type. See also Figure S2B.
Structure  , DOI: ( /j.str )
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4. Figure 2
Effects of smc3(T1185M) and smc1(S1189A) Mutations on Cohesin Functions
(A) ATPase assay. Hydrolysis of [γ-32P]ATP was monitored using thin-layer chromatography. Smc1 (K39I) is a Walker A mutant defective in ATP hydrolysis and was used as a control. Coomassie-stained gels of Smc1/Smc3 and C-Scc1 preparations, respectively, are shown on the left.
(B) smc3(T1185M) and smc1(S1189A) reduce the interaction of Wpl1 with cohesin. Scc1-TAP was pulled down with immunoglobulin G Sepharose and the presence of Wpl1-Myc on the beads was analyzed by western blot. Increasing volumes (1×, 2×, 3×) of each sample were loaded. Co-immunoprecipitation experiments were repeated three times with similar results.
(C) smc3(T1185M) reduces the association of cohesin with DNA. ChIP-qPCR was performed using nocodazole-arrested yeast. Scc1-Pk6 was immunoprecipitated and binding of cohesin to chromosomes was quantified. ChIP-qPCR experiments were repeated several times with similar results. See also Figure S2C.
Structure  , DOI: ( /j.str )
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5. Figure 3 Distinct Features of the Smc1 and Smc3 Interfaces
(A) Simulation-refined structure of the Smc1/Smc3 heterodimer. The dimeric interface comprises two distinct sections labeled as the Smc1 interface and the Smc3 interface. ATP is shown in CPK wireframe format. Regions of interaction between the two protein chains are colored as labeled. See also Figure S3.
(B) Details of the Smc3 interface. The key interactions of the Smc3 interface are highlighted by arrows. Crucial contacts comprise the salt bridge Smc1-D1160 to Smc3-R1187, the amide-π contact between Smc1-A1162 and Smc3-F1186, and the hydrogen bond between Smc1-D1164 and Smc3-S33. Mg2+ is depicted as a solid sphere, and some helices are omitted for clarity.
(C) Time course of the opening of the Smc1/Smc3 interfaces upon ATP hydrolysis. In the ATP/ATP non-hydrolyzed state and the ATP/ADP state representing hydrolysis only at the Smc3 active site, the heads remain together during the course of the simulation. Conversely, the interface opens in the case of simulated hydrolysis at the Smc1 active site (ADP/ATP) and in the dually hydrolyzed state (ADP/ADP). It should be noted that interface dissociation is more pronounced in the doubly hydrolyzed state.
See also Movie S1.
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6. Figure 4 Mutations that Rescue Δeco1 Disrupt the Smc3 Interface
(A) Smc1 and Smc3 mutations that rescue Δeco1. Mutations that rescue Δeco1 are predominantly located at the Smc3 interface and are indicated in green. The only mutation that is located closer to the Smc1 interface is Smc1-S1189A, but simulations showed that this also primarily affects the Smc3 interface as it abolishes hydrogen bonding with the overlaying parallel loop segment indicated in blue. See also Figure S4A.
(B) The Smc1 D-loop and Smc3 H-loop amino acids employed in the crosslinking experiments.
(C) The Smc1 and Smc3 heads can be crosslinked at Smc3-F1186. Strains expressing photo-crosslinkable Smc3 (T1184∗BPA, F1186∗BPA, R1187∗BPA, or T1188∗BPA) from a plasmid were UV-irradiated and Smc1-Myc was detected by western blot. The Smc1/Smc3 dimers can be strongly crosslinked at Smc3-F1186 and weakly at Smc3-T1184 and R1187, but not at Smc3-T1188. See also Figure S4B.
(D) smc3(T1185M) but not smc3(KK112,113QQ) adversely affects the interaction between the Smc1 and Smc3 heads. Strains expressing photo-crosslinkable Smc3 (F1186∗BPA), Smc3 (T1185M, F1186∗BPA), or Smc3 (KK112,113QQ, F1186∗BPA) from a plasmid were UV-irradiated and Smc1-Myc was detected by western blot.
(E) smc1(D1164E) and smc1(S1189A) impede Smc1/Smc3 head engagement. Strains expressing both Smc3 (F1186∗BPA) and Smc1 (WT)-Myc, Smc1 (D1164E)-Myc, or Smc1 (S1189A)-Myc from plasmids were UV-irradiated and Smc1-Myc was detected by western blot.
Structure  , DOI: ( /j.str )
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