DNA passes through cohesin’s hinge as well as its Smc3–kleisin interface
- Department of Biochemistry, University of Oxford, United Kingdom
Figures
Figure 1 with 1 supplement
Covalent closure of cohesin’s interfaces.
(A) The cohesin ring and fusion variants. Covalently sealed interfaces are linked in black and are indicated with arrows. (B) Coomassie stain of purified cohesin with either the Smc3/Scc1 (S3 …
Figure 1—figure supplement 1
Covalent closure of cohesin’s interfaces.
(A) Top: the polypeptide sequences used to fuse Scc1 to Smc1 and Smc3. Bottom: the polypeptide sequences of the spytag and spycatcher inserted into Smc1 (after residue L597) and Smc3 (after residue …
Figure 2 with 1 supplement
DNA passes through cohesin’s hinge and Smc3/Scc1 interfaces.
(A) Schematic of the in vitro DNA entrapment assay. (B) SMC–kleisin (S–K) ring. (C) DNA entrapment comparing WT cohesin (6C) with the Smc3–Scc1 (S3), Scc1–Smc1 (S1), or hinge (Hinge) fusion proteins …
Figure 2—figure supplement 1
DNA entrapment by SMC–kleisin (S–K) rings.
DNA entrapment for WT cohesin (6C) in the presence or absence of Scc2 and Scc3 after 40 min.
Figure 3 with 2 supplements
DNA passes through cohesin’s ATPase domains.
(A) Crosslinking Scc2 to the Smc1 head in the presence or absence of ATP and DNA. n = 3. (B) Crosslinking Scc2 to the Smc3 head in the presence or absence of ATP and DNA. n = 3. (C) Crosslinking …
Figure 3—figure supplement 1
DNA passes through cohesin’s ATPase domains.
(A) Left panel: Scc2–Smc1 head crosslinking pair. Middle panel: Scc2–Smc3 head crosslinking pair. Right panel: Scc2–Smc3 neck crosslinking pair. (B) DNA could enter the E-S compartment by going …
Figure 3—figure supplement 2
Entrapment in the E-S and E-K compartments occurs simultaneously.
(A) Models showing that cleavage of both the E-S and E-K compartments is required to release DNA that has passed ‘up’ through the heads. (B) A TEV protease site is located in the linker between the …
Figure 4
Models for DNA entry.
(A) Model for DNA passage through the hinge. DNA passes ‘up’ through the ATPase heads and binds to Scc2 (purple). The SMC heads engage in the presence of ATP, and DNA is then clamped between Scc2 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant DNA reagent | pACEbac1 Smc1 Smc3 Scc1-StrepII | This Study | pJC 93 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Smc1 G22C K639C Smc3 E570C S1043C Scc1-StrepII A547C | This Study | pJC 95 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Smc1 K639C N1192C Smc3 E570C R1222C Scc1-StrepII | This Study | pJC 153 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Smc1 G22C N1192C Smc3 S1043C R1222C Scc1-StrepII A547C | This Study | pJC 154 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Smc1 G22C K639C Smc3Scc1-StrepII E570C A547C | This Study | pJC 127 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Smc1 G22C N1192C Smc3 S1043C R1222C Scc1-StrepII A547C | This Study | pJC 123 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Smc1 G22C L597ST Smc3 S1043C S606SC Scc1-StrepII A547C | This Study | pJC 106 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Smc3Scc1Smc1-StrepII E570C K639C | This Study | pJC 132 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Smc1 G22C L597ST Smc3Scc1-StrepII S606SC A547C | This Study | pJC 129 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Smc3 S1043C S606SC Scc1Smc1-StrepII L597ST | This Study | pJC 124 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Smc3Scc1Smc1-StrepII S606SC TEV R1222C L597ST N1192C | This Study | pJC 135 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1i Smc1-His E1102C Smc3 Scc1-StrepII | This Study | pJC 151 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1i Smc1 Smc3 S72C Scc1-StrepII | This Study | pJC 100 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1i Smc3 K1004C Smc1 _Scc1-StrepII | This Study | pJC 101 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1i Smc1 Smc3 S72C K1004C Scc1-StrepII | This Study | pJC 102 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1i Smc1 E1102C N1192C Smc3 K1004C R1222C Scc1-StrepII | This Study | pJC 105 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Scc2133-1493-StrepII | Collier et al., 2020 | pJC 69 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Scc2133-1493-StrepII T1281C | This Study | pJC 75 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Scc2133-1493-StrepII E819C | This Study | pJC 77 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Scc2133-1493-StrepII D369C | This Study | pJC 76 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Scc2133-1493-StrepII D369C E819C | This Study | pJC 78 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 Scc2133-1493-StrepII D369C T1281C | This Study | pJC 80 | Plasmid obtainable from Nasmyth Lab |
| Recombinant DNA reagent | pACEbac1 StrepII-Scc3 | Collier et al., 2020 | pJC 84 | Plasmid obtainable from Nasmyth Lab |
| Chemical compound, drug | ATP | Sigma | Cat # 11140965001 | |
| Chemical compound, drug | BMOE | ThermoFisher | Cat # 22323 | |
| Chemical compound, drug | HRV 3 C protease | Pierce | Cat # 88946 | |
| Chemical compound, drug | TEV protease | Invitrogen | Cat # 12575023 | |
| Chemical compound, drug | EnzChek phosphate assay kit | Invitrogen | Cat # E6646 |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/80310/elife-80310-mdarchecklist1-v1.pdf
-
Source data 1
All source data from the study.
- https://cdn.elifesciences.org/articles/80310/elife-80310-data1-v1.zip
