Communication between distinct subunit interfaces of the cohesin complex promotes its topological entrapment of DNA
- University of California, Berkeley, United States
Figures
Figure 1
Cohesin structure.
(A) Cartoon showing the cohesin complex and Pds5p. The three interfaces that can be chemically crosslinked to trap DNA within the Smc1p-Smc3p-Mcd1p trimer are marked by arrows. These are 1) Smc3p …
Figure 2
Cohesin regulators are required fusion cohesin function.
(A-B) Haploid strains with normal cohesin or fusion were grown to saturation, then plated at 10-fold serial dilution onto YPD alone or containing auxin (750 μM) and incubated at 23°C for 3 days. (A) …
Figure 3 with 2 supplements
Fusion cohesin requires cohesin regulators for sister chromatid cohesion and cohesin binding to DNA.
Haploids strains with normal cohesin or fusion cohesin from Figure 2 were grown to mid-log phase then arrested in G1 using α factor, auxin added to induce loss of AID-tagged proteins then released …
Figure 3—figure supplement 1
Characterizing the effect of Scc2p-AID or Scc3p-AID depletion in normal and fusion cohesin strains.
(A) Schematic of regimen used to synchronously arrest cells in mid-M phase. (B–D) Strains with normal cohesin or fusion cohesin alone or bearing Scc2p-AID or Scc3p-AID from Figure 2A were …
Figure 3—figure supplement 2
Characterizing the effects of Pds5p-AID depletion in normal and fusion cohesin strains.
Strains with normal cohesin or fusion cohesin alone or bearing Pds5p-AID from Figure 2B were synchronously arrested in mid-M then processed to generate data presented in described in Figure 3B and D.…
Figure 4 with 1 supplement
Fusion cohesin with the smc3 K112R K113R mutations are viable but have defects in growth defects in both growth and sister chromatid cohesion.
(A–B) Fusion cohesion bearing smc3-K112R, K113R (RR) mutations are viable but cold sensitive and benomyl sensitive. Haploid strains with wild-type fusion cohesin (VG3940-2D) or RR fusion (VG3930-5C) …
Figure 4—figure supplement 1
Characterization of fusion wild-type and smc3-K112R K113R (RR) mutations.
Strains with fusion cohesin or RR fusion cohesin in Figure 4 were grown treated as described. (A) FACS to confirm arrest of cells. (B) Smc3p-Mcd1p fusion levels in mid-M arrested cells. Protein …
Figure 5 with 1 supplement
Smc3p coiled-coil and Mcd1p NHD interface residues are required for cohesin function and integrity.
(A) Viability loss of Smc3p interface mutant strains. Haploid SMC3-AID strain alone (VG3651-3D) or containing either wild-type (WT; BRY474), smc3-I1026R (VG3905-7A) or smc3-L1029R (BRY492) were …
Figure 5—figure supplement 1
FACS analysis showing cell cycle arrest of normal cohesin strains bearing interface mutations.
Strains with normal cohesin alone or bearing interface mutants were synchronously arrested in mid-M as described in Figure 3. (A) smc3 interface mutants in normal cohesin strains from Figure 5. (B) m…
Figure 6 with 3 supplements
Fusion cohesin requires Smc3p coiled coil residues for function.
(A) Viability loss of Smc3p coiled coil mutant fusion strains. Haploid SMC3-AID strain alone (VG3651-3D) or containing fusion cohesin, either wild-type (WT; VG3694-7C), smc3-I1026R (VG3908-17B) or sm…
Figure 6—figure supplement 1
Confirming cell cycle arrest of fusion cohesin bearing smc3 interface mutants.
Strains with fusion cohesin alone or bearing smc3 interface mutants from Figure 6 were synchronously arrested in mid-M as described. FACS analysis was used to confirm arrest.
Figure 6—figure supplement 2
Western blot to assess the effect of smc3-L1029R interface mutant on cohesin levels.
Haploid normal cohesin strains bearing Smc3p-3V5-AID alone (VG3651-3D) or containing either Smc3p-6HA (VG3943-1C) or Smc3p-6HAL1029R (VG3944-3D) were grown asynchronously, auxin added and cells …
Figure 6—figure supplement 3
Fusion wild-type and fusion L1029 mutant coimmunoprecipitate with Scc2p and Scc3p equally well.
(A) Fusion wild-type and L1029R mutant cohesin both co-IP with Scc2p. Haploid strain Scc2p-6MYC, Smc3p-3V5-AID bearing wild-type fusion cohesin (VG3952-14C) or fusion L1029R mutant (VG3953-17D) were …
Figure 7 with 1 supplement
Fusion cohesin requires Mcd1p NHD residues for function.
(A) Viability loss of Mcd1p NHD mutant fusion strains. Haploid MCD1-AID strain alone (VG3902-3A) or containing fusion cohesin, either wild-type (WT; VG3937-2C), mcd1-L75K (VG3938-3A) or mcd1-L89K …
Figure 7—figure supplement 1
Confirming cell cycle arrest of fusion cohesin bearing mcd1 NHD interface mutants.
Strains with fusion cohesin alone or bearing mcd1 interface mutants from Figure 7 were synchronously arrested in mid-M as described. FACS analysis was used to confirm arrest.
Figure 8
Model for how the Smc3p Mcd1p interface regulates the hinge dimer interface to control cohesin DNA binding.
(A) Left side: cohesin complex in an open ring conformation. Middle top: cohesin loader Scc2p/Scc4p binding at the Smc3p Mcd1p interface (not shown) triggers a conformation change that opens the …
Tables
Key resources table
| Reagent type or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (S. cerevisiae) | NCBITaxon:4932 | this paper | Yeast strains | Supplementary file 1 |
| qPCR primers | DNA primer | IDT | Supplementary file 2 | |
| Antibody | Rabbit polyclonal Anti-Mcd1p | V. Guacci (via Covance) | RbαMcd1p (555) | WB (1:10,000) ChIP (1:1,000) |
| Antibody | Rabbit polyclonal Anti-Pds5p | V. Guacci (via Covance) | RbαPds5p (556) | WB (1:20,000) ChIP (1:1,000) |
| Antibody | Mouse monoclonal Anti-MYC | Roche | MαMYC (9E10) Cat#116666006001 | WB (1:10,000) IP (1:667) |
| Antibody | Mouse monoclonal Anti-HA | Roche | MαHA (12CA5) Cat#11667203001 | WB (1:10,000) |
| Antibody | Mouse monoclonal Anti-V5 | Invitrogen | MαV5 Cat# 46–0705 | WB (1:10,000) |
| Antibody | Goat polyclonal HRP Anti-rabbit | Biorad | Cat# 170–6515 | WB (1:10,000) |
| Antibody | Goat polyclonal HRP Anti-mouse | Biorad | Cat# 170–6516 | WB (1:10,000) |
| Antibody | Rabbit polyclonal Anti-Tub2p | P. Meluh (via Covance) | RbαTUB2 | WB (1:40,000) |
| Dynabeads | Protein A | Invitrogen | Ref# 10002D | IP: Use 50 ul/IP |
| Dynabeads | Protein G | Invitrogen | Ref# 10004D | IP: Use 50 ul/IP |
| Chemical compound | Auxin (3-indole acetic acid) | Sigma | Cat# C9911 | 750 μM for plates 500 μM for liquid |
| Chemical compound | Alpha factor | Sigma | αF (αFactor) Cat# T6901 | |
| Chemical compound | Nocodazole | Sigma | Nz Cat# M1404 |
Additional files
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Supplementary file 1
Yeast strains.
- https://doi.org/10.7554/eLife.46347.018
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Supplementary file 2
Primers used for chromatin immunoprecipitation (ChIP).
- https://doi.org/10.7554/eLife.46347.019
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Transparent reporting form
- https://doi.org/10.7554/eLife.46347.020
