Transport of DNA within cohesin involves clamping on top of engaged heads by Scc2 and entrapment within the ring by Scc3
- Department of Biochemistry, University of Oxford, United Kingdom
- MRC Laboratory of Molecular Biology, United Kingdom
Figures
Figure 1 with 1 supplement
SMC-kleisin (S–K) rings entrap circular DNA in vitro.
(A) Cohesin’s different compartments and the position of cysteine pairs used in our crosslinking studies. (B) BMOE-induced crosslinking of S-K rings with cysteine pairs in the specified interfaces. …
Figure 1—figure supplement 1
Related to Figure 1.
(A) Left hand panel - Coomassie strain of cohesin trimers. Individual subunits were then probed for by western blotting. Right hand panel – Coomassie stain of Scc2C and Scc3. (B) Left hand panel - …
Figure 2
Entrapment within S-K rings requires both Scc2 and Scc3, ATP binding to Smc3, and is stimulated by ATP hydrolysis.
(A) Entrapment of DNA in S-K rings in the presence of Scc3, and the presence or absence of Scc2, or (B) the presence of Scc2, and the presence or absence of Scc3 (*=damaged open circular DNA). (C) …
Figure 3 with 1 supplement
DNA binding to Scc3 is required for its entrapment by S-K rings.
(A) Structure of S. cerevisiae Scc3 (orange) protein in complex with a fragment of Scc1 (green) (PDB 6H8Q). Labelled are the six residues within the DNA binding groove of Scc3 that were mutated to …
Figure 3—figure supplement 1
Related to Figure 3.
(A) EMSA comparing the ability of Scc3 to bind dsDNA against Scc3-Scc1269-451 complexes, or (B) of Scc3-3E(1)-Scc1269-451 (Scc3K224E K225E R226E) against Scc3-3E(2)-Scc1269-451 (Scc3K423E K513E …
Figure 4 with 1 supplement
DNA binding to Scc2 facilitates entrapment by S-K rings.
(A) EMSA comparing the ability of Scc2 and Scc2-Scc1150-298 complexes to bind dsDNA. (B) S. cerevisiae Scc2 from the cryo-EM structure (Figure 8) with the four resides within the putative DNA …
Figure 4—figure supplement 1
Related to Figure 4.
(A) EMSA comparing the ability of full length Scc2/4 and Scc2/4-Scc1150-298 complexes to bind dsDNA or (B) Comparing Scc2-2E(1) (Scc2S717E K721E) and Scc2-2E(2) (Scc2K788E H789E). (C) Average …
Figure 5 with 2 supplements
Rapid DNA entrapment in E-S and E-K compartments.
(A) Entrapment of DNA in E-S/E-K compartments in the presence of Scc2 and Scc3, and the presence or absence of ATP, incubated for 40 min (*=damaged open circular DNA). (B) DNA entrapment in S-K …
Figure 5—figure supplement 1
DNA is never entrapped in J-S and only rarely in J-K compartments.
(A) BMOE crosslinking of J-K compartments, or (B) of J-S compartments, with cysteine pairs in the designated interfaces. CC = circular cohesin. (C) Crosslinking of J-S or J-K compartments in the …
Figure 5—figure supplement 2
Circularisation of the E-S and E-K compartments and E- and J-state crosslinking under different conditions.
(A) BMOE crosslinking of cohesin containing a cysteine pair for E head association, in the presence of ATP, DNA, or Scc2 in various combinations and in the presence of 1 mM MgCl2. (B) Crosslinking …
Figure 6
E-S/E-K entrapment leads to dissociation of the coiled coil around the joint.
(A) Scheme showing the location of the joint cysteine pair and how head engagement could lead to different degrees of coiled coil dissociation and sub-compartment formation. (B) BMOE crosslinking of …
Figure 7
Cryo-EM of cohesin clamping DNA in the E-S/E-K state.
(A) Cryo-EM map of 40 bp DNA clamped by Scc2- and ATP-bound cohesin EQEQ trimer at 3.4 Å resolution. Both front and side views are coloured by subunit. (B) Same complex as shown in A but bound to ~1.…
Figure 8 with 1 supplement
Molecular interactions in the E-S/E-K state.
(A) Cartoon representation of the refined atomic model of cohesin’s clamped (E-S/E-K) state based on the 3.4 Å resolution cryo-EM map (Figure 7A, same orientation and colours, Supplementary file 1). …
Figure 8—figure supplement 1
Comparison of cryo-EM structures.
(A) Cohesin tetramer (yeast, this study), (B) pentamer (human, 6WG3) (Shi et al., 2020) and (C) condensin (yeast, 6YVU) (Lee et al., 2020) are shown in two orientations (same or similar to Figures 7A…
Figure 9
Potential mechanisms for Scc2-driven E-S/E-K entrapment and subsequent S-K ring entrapment.
(A) ES/EK entrapment by DNA passing through open heads, or (B) through a DNA loop being inserted. (C) Topological isomerism between A and B. (D) ES/EK entrapment due to two distinct populations. (E) …
Videos
Video 1
A model for the formation of the clamped E-S/E-K state of cohesin.
According to this model cohesin transitions from a putative ‘bridged state’ (modelled on the same state of yeast apo condensin as observed by cryo-EM) (Lee et al., 2020) in which Scc2, analogous to …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (S. cerevisiae) | MATa ura::ADH1 promoter-OsTIR1-9myc::URA3 Scc3-PK3-aid::KanMX4 SCC1-HA3::HIS3 | This study | KN20783 | |
| Strain, strain background (S. cerevisiae) | MATa Scc3-PK3-aid::KanMX4 SCC1-HA3::HIS3 | This study | KN20785 | |
| Strain, strain background (S. cerevisiae) | MATa/alpha scc3::NatMX4/WT | This study | KN21079 | |
| Strain, strain background (S. cerevisiae) | MATa/alpha scc3::NatMX4/WT, leu::Scc3-HA3::LEU | This study | KN21273 | |
| Strain, strain background (S. cerevisiae) | MATa Scc1-PK9::KanMX scc2-45::natMX (L545P D575G) | This study | KN22390 | |
| Strain, strain background (C. glabrata) | MATa, SCC1-PK9::NATMX4 | Petela et al., 2018 | KN23308 | |
| Strain, strain background (S. cerevisiae) | MATa Scc1-PK9::KanMX scc2-45::natMX (L545P D575G) lys2::Scc2-HyGMX | This study | KN24185 | |
| Strain, strain background (C. glabrata) | MATa, SCC1-HA3::NATMX4 | Petela et al., 2018 | KN25532 | |
| Strain, strain background (S. cerevisiae) | MATa Scc1-PK9::KanMX scc2-45::natMX (L545P D575G) LYS2::Scc2(S717L,K721E)-HygMX | This study | KN27010 | |
| Strain, strain background (S. cerevisiae) | MATa/alpha scc3::NatMX4/WT, leu::Scc3 (K224E, K225E, R226E)-HA3::LEU | This study | KN27539 | |
| Strain, strain background (S. cerevisiae) | MATa scc3::NatMX4, Scc1-PK6::TRP1, leu::Scc3-HA3::LEU | This study | KN27542 | |
| Strain, strain background (S. cerevisiae) | MAT alpha scc3::NatMX4, Scc1-PK6::TRP1, leu::Scc3 (K224E, K225E, R226E)-HA3::LEU | This study | KN27547 | |
| Strain, strain background (S. cerevisiae) | MATa/alpha scc3::NatMX4/WT, leu::Scc3 (K423E, K513E, K520E)-HA3::LEU | This study | KN27696 | |
| Strain, strain background (S. cerevisiae) | MATa scc3::NatMX4, Scc1-PK6::TRP1, leu::Scc3 (K423E, K513E, K520E)-HA3::LEU | This study | KN27697 | |
| Strain, strain background (S. cerevisiae) | MATa/alpha scc3::NatMX4/WT, leu::Scc3 (K224E, K225E, R226E, K423E, K513E, K520E)-HA3::LEU | This study | KN27763 | |
| Strain, strain background (S. cerevisiae) | MATa ura::ADH1promoter-OsTIR1-9myc::URA3, Scc3-PK3-aid::KanMX4, leu::Scc3-HA3::LEU | This study | KN27796 | |
| Strain, strain background (S. cerevisiae) | MATa ura::ADH1 promoter-OsTIR1-9myc::URA3, Scc3-PK3-aid::KanMX4 leu::Scc3 (K224E, K225E, R226E, K423E, K513E, K520E)-HA3::LEU | This study | KN27802 | |
| Strain, strain background (S. cerevisiae) | MATa ura::ADH1promoter-OsTIR1-9myc::URA3, Scc3-HA3-aid::KanMX4, Scc1-PK6::TRP1, leu::Scc3 (K224E, K225E, R226E, K423E, K513E, K520E)-HA3::LEU | This study | KN27804 | |
| Strain, strain background (S. cerevisiae) | MATa ura::ADH1promoter-OsTIR1-9myc::URA3, Scc3-HA3-aid::KanMX4, Scc1-PK6::TRP1, leu::Scc3-HA3::LEU | This study | KN27821 | |
| Strain, strain background (S. cerevisiae) | MATa ura::ADH1promoter-OsTIR1-9myc::URA3, Scc3-HA3-aid::KanMX4, leu::Scc3-HA3::LEU, Scc2-PK9::NatMX | This study | KN28075 | |
| Strain, strain background (S. cerevisiae) | MATa ura::ADH1 promoter-OsTIR1-9myc::URA3, Scc3-HA3-aid::KanMX4, Scc2-PK9::NatMX, leu::Scc3 (K224E, K225E, R226E, K423E, K513E, K520E)-HA3::LEU | This study | KN28287 | |
| Strain, strain background (S. frugiperda) | Sf9 insect cells | ThermoFisher | Cat# 11496015 | |
| Antibody | Anti-His (mouse) | Sigma | Cat# SAB1305538-400UL | 1:2000 |
| Antibody | Anti-mouse HRP | ThermoFisher | Cat# 62–6520 | 1:5000 |
| Antibody | Anti-Smc3 (mouse) | Bethyl Laboratories | Cat# A300-060A | 1:500 |
| Antibody | Anti-Strep HRP | iba | Cat# 2-1502-001 | 1:4000 |
| Recombinant DNA reagent | pACEbac1 SMC1-His | This Study | ||
| Recombinant DNA reagent | pACEbac1 SMC3 | This Study | ||
| Recombinant DNA reagent | pACEbac1 SMC1-His SMC3 | This Study | ||
| Recombinant DNA reagent | pACEbac1 Scc2133-1493-2xStrepII | This Study | ||
| Recombinant DNA reagent | pACEbac1 SCC2-2xStrepII | This Study | ||
| Recombinant DNA reagent | pACEbac1 2xStrepII-Scc2151-1493 | This Study | ||
| Recombinant DNA reagent | pACEbac1 2xStrepII-SCC3 | This Study | ||
| Recombinant DNA reagent | pIDC SCC1-2xStrepll | This Study | ||
| Recombinant DNA reagent | pIDC Scc1269-451-2xStrepII | This Study | ||
| Recombinant DNA reagent | pIDC Scc1150-298-2xStrepII | This Study | ||
| Recombinant DNA reagent | pIDC His-SCC4 | This Study | ||
| Chemical compound, drug | ATP Lithium Salt | Sigma | Cat# 11140965001 | |
| Chemical compound, drug | Bismaleimidoethane (BMOE) | ThermoFisher | Cat# 22323 | |
| Chemical compound, drug | Complete EDTA free protease inhibitor cocktail | Roche | Cat# 4693132001 | |
| Chemical compound, drug | Cre Recombinase | New England Biolabs | Cat# M0298S | |
| Chemical compound, drug | Desthiobiotin | Fisher Scientific | Cat# 12753064 | |
| Chemical compound, drug | EtBr | ThermoFisher | Cat# 15585011 | |
| Chemical compound, drug | Fetal Bovine Serum | Sigma | Cat# 12303C | |
| Chemical compound, drug | FuGENE HD Transfection reagent | Promega | Cat# E2311 | |
| Chemical compound, drug | Gibson Assembly Mix | New England Biolabs | Cat# E2611L | |
| Chemical compound, drug | Immobilon Western ECL | Millipore | Cat# WBLKS0500 | |
| Chemical compound, drug | NuPAGE 3–8% Tris-Acetate Protein Gels | ThermoFisher | Cat# EA0378BOX | |
| Chemical compound, drug | PMSF | Sigma | Cat# 329-98-6 | |
| Chemical compound, drug | Quick Coomassie Stain | Generon | Cat# GEN-QC-STAIN | |
| Chemical compound, drug | RNase A | Roche | Cat# 10109169001 | |
| Chemical compound, drug | Sf900 II SFM | ThermoFisher | Cat# 10902104 | |
| Chemical compound, drug | Supernuclease | SinoBiological | Cat# SSNP01 | |
| Chemical compound, drug | TCEP | ThermoFisher | Cat# 20490 | |
| Chemical compound, drug | 4xLDS | ThermoFisher | Cat# NP0007 | |
| Commercial assay or kit | HiLoad 16/60 Superdex 200 | GE Healthcare | Cat# GE28-9893-35 | |
| Commercial assay or kit | HiSpeed Plasmid Maxi Kit | Qiagen | Cat# 12663 | |
| Commercial assay or kit | HiTrap Q HP | GE Healthcare | Cat# GE29-0513-25 | |
| Commercial assay or kit | StrepTrap HP | Fisher Scientific | Cat# 11540654 | |
| Commercial assay or kit | Superose 6 Increase 10/300 GL | VWR | Cat# 29-0915-96 | |
| Commercial assay or kit | EnzChek phosphate assay kit | Invitrogen | Cat# E6646 | |
| Software, algorithm | RELION 3.1 | doi:10.1016/j.jsb.2012.09.006 | ||
| Software, algorithm | CtfFind4 | doi:10.1016/j.jsb.2015.08.008 | ||
| Software, algorithm | Warp | doi:10.1038/s41592-019-0580-y | ||
| Software, algorithm | CrYOLO 1.5 | doi:10.1038/s42003-019-0437-z | ||
| Software, algorithm | Chimera | https://www.cgl.ucsf.edu/chimera/ | ||
| Software, algorithm | ChimeraX 1.0 | https://www.cgl.ucsf.edu/chimerax/ | ||
| Software, algorithm | COOT | doi:10.1107/S0907444910007493 | ||
| Software, algorithm | MAIN | doi:10.1107/S0907444913008408 | ||
| Software, algorithm | Phenix.real_space_refinement | doi:10.1107/S2059798318006551 | ||
| Software, algorithm | PYMOL 2 | https://pymol.org/2/ | ||
| Software, algorithm | SWISS-MODEL | https://swissmodel.expasy.org | ||
| Other | Quantifoil Au 2/2 holely carbon 200 mesh cryoEM grids | Quantifoil GmbH | ||
| Other | Ultrafoil 2/2 holely gold 200 mesh cryoEM grids | Quantifoil GmbH |
