Scc2 counteracts a Wapl-independent mechanism that releases cohesin from chromosomes during G1
- University of Oxford, United Kingdom
- Centre de Biologie Intégrative (CBI), Université de Toulouse, France
- University of Sheffield, United Kingdom
Figures
Figure 1 with 1 supplement
A Wapl-independent activity releases cohesin from chromosomes in G1 cells.
(A) Cohesin’s association with DNA is regulated by two opposing activities: Scc2-Scc4 complex loads cohesin onto DNA, while Pds5, Scc3 and Wapl constitute the releasing activity that releases cohesin from DNA by opening the Smc3-Scc1 interface. Mutations in Scc3 (scc3K404E) Pds5 (pds5S81R) and deletion of the WAPL gene abrogate Wapl mediated releasing activity and lead to cohesin’s stable association with DNA. (B) Schematic of the mini-chromosome IP assay: 6C strain (K23889) with cysteine pairs at all three ring subunit interfaces (2C Smc3: E570C S1043C, 2C Smc1: G22C K639C and 2C Scc1 C56 A547C) and carrying a 2.3 kb circular mini-chromosome was subjected to in vivo crosslinking with BMOE. DNAs associated with cohesin immune-precipitates (Scc1-PK6) were denatured with SDS and separated by agarose gel electrophoresis. Southern blotting reveals two forms of DNA unique to cells treated with BMOE: CMs (cohesin entrapping individual mini-chromosomes) and CDs (cohesin entrapping a pair of sister mini-chromosomes). (C) WT (K23972) and scc2-45 (K25238) 6C strains were arrested in late G1 by overexpression of nondegradable Sic1 at 25°C as described in Materials and Methods. The cultures were shifted to 37°C for 20 min, aliquots drawn before (0) and after (20) temperature shift (to inactivate Scc2) were subjected to mini-chromosome IP. (D) scc3K404E (K25313) and scc3K404E scc2-45 (K25316) 6C strains were arrested in late G1. The cultures were shifted to 37°C for 20 min, aliquots drawn before (0) and after (20) temperature shift (to inactivate Scc2) were subjected to mini-chromosome IP. Also see S1B. (E) pds5S81R (K25311) and pds5S81R scc2-45 (K25312) 6C strains were arrested in late G1. The cultures were shifted to 37°C for 20 min, aliquots drawn before (0) and after (20) temperature shift (to inactivate Scc2) were subjected to mini-chromosome IP. (F) scc3K404E (K25313) and scc3K404E scc2-45 (K25316) strains were arrested in late G1. The cultures were shifted to 37°C for 20 min, aliquots drawn before (0) and after (20) temperature shift (to inactivate Scc2) were analysed by Calibrated-ChIP- sequencing (Scc1-PK). Cohesin profile along chromosome four is shown. Also see Figure 1—figure supplement 1. (G) Even in cells that lack Wapl mediated releasing activity, inactivation of Scc2 in G1 cells leads to release of DNA entrapped within cohesin rings. This suggests that an activity that is Wapl-independent is capable of releasing cohesin from DNA.
Figure 1—figure supplement 1
A Wapl-independent activity releases cohesin from chromosomes in G1 cells.
(A) Data from Figure 1F is plotted to show the percentage of cohesin that remains on DNA upon Scc2 inactivation (after temperature shift) along the entire chromosome 4. The median cohesin level along the entire chromosome 4 (dotted line) is marked with arrowheads. (B) FACS analysis of the cultures described in Figure 1D (bottom two) and Figure 1—figure supplement 1F (top two). (C) Strain containing smc3R1008I mutation was crossed with an eco1Δ waplΔ strain and the resultant diploid analysed by tetrad dissection. The spores with eco1Δ waplΔ and eco1Δ waplΔ smc3R1008I are highlighted. (D) FACS analysis of strains grown in Figure 2A. (E) FACS analysis of strains grown in Figure 2B. (F) scc3K404E (K25313) and scc3K404E scc2-45 (K25316) 6C strains were arrested in G2 with nocadazole as described in Methods. The cultures were shifted to 37°C for 20 min, aliquots drawn before (0) and after (20) temperature shift (to inactivate Scc2) were subjected to mini-chromosome IP. Also see Figure 1—figure supplement 1B. (G) The cultures from experiment described in Figure 2A were analysed by western blotting with the indicated antibodies. (H) The Scc2-3XmAID strain described in Figure 2G was analysed by western blotting with anti-mAID antibody and anti PK antibody before and after auxin addition. (I) The Scc2-3XmAID strain described in Figure 2H was analysed by western blotting with anti-mAID antibody and anti PK antibody before and after auxin addition.
Figure 2
The Wapl-independent activity that releases cohesin from chromosomes is active only in G1 cells.
(A) waplΔ (K22296) and waplΔ scc2-45 (K22294) strains were arrested in late G1 at 25°C and subjected to temperature shift to 37°C for 20 min. 0- and 20 min samples were analysed by calibrated ChIP-sequencing (Scc1-PK6) as detailed in Materials and Methods. Cohesin ChIP profiles along chromosomes four is shown. Also see Figure 1—figure supplement 1D. (B) waplΔ (K22296) and waplΔ scc2-45 (K22294) strains were arrested in G2 with nocadazole at 25°C and subjected to temperature shift to 37°C for 20 min. 0- and 20 min samples were analysed by calibrated ChIP-sequencing (Scc1-PK6). Cohesin ChIP profiles along chromosomes four is shown. Also see Figure 1—figure supplement 1E. (C) Data form (A) is plotted to show the ratio of average cohesin levels 60 kb on either side of all 16 centromeres before and 20 min after the temperature shift. (D) Data form (B) is plotted to show the ratio of average cohesin levels 60 kb on either side of all 16 centromeres before and 20 min after the temperature shift. (E and F) waplΔ (K20891) and scc2-3XmAID waplΔ (K26831) were arrested in either late G1 or G2 and treated with auxin (IAA) for 60 min (to degrade Scc2) and subjected to Cal-ChIP-Seq. Samples drawn before (0 min) and after (60 min) auxin addition were analysed by calibrated ChIP-sequencing (Scc1-PK6). Cohesin ChIP profiles along chromosomes four is shown. Also see Figure 1—figure supplement 1H and I. (G and H) Data form (E and F respectively) are plotted to show the ratio of average cohesin levels 60 kb on either side of all 16 centromeres before and 60 min after auxin addition (I and J) The Wapl-independent activity that releases cohesin from DNA is active only in G1 (I) and not in mitotic cells (J).
Figure 3 with 1 supplement
Pds5 is not required for the Wapl-independent release and Scc2 does not require Scc4 to inhibit the Wapl-independent release.
(A) waplΔ (K27569) and waplΔ scc4-4 (K27570) strains were arrested in late G1 at 25°C and subjected to temperature shift to 37°C for 30 min. Ratio of average cohesin levels before and 30 min after the temperature shift is plotted. Also see S2A and D. (B) Association with Scc4 is not required for Scc2 to block the Wapl-independent release. Scc2 actively inhibits release in G1. (C) pds5-AID (K26415) and pds5-AID scc2-45 (K26414) strains were arrested in G1 with α-factor and released into sic1(late G1) arrest in the presence of auxin (IAA) and subjected to temperature shift and Cal-ChIP-Seq. Ratio of average cohesin levels before and temperature shift is plotted. Also see Figure 3—figure supplement 1B and C.
Figure 3—figure supplement 1
Pds5 is not required for the Wapl-independent release and Scc2 does not require Scc4 to inhibit the Wapl-independent release.
(A) Strain expressing Scc2-PK (K25738) and scc4-4 Scc2-PK (K28081) were arrested in G1 with alpha factor at 25°C and released into a later G1 arrest (sic1-9m overexpression) at 37°C and analysed by western blotting with indicated antibodies. (B) Strain (K26415) described in Figure 3A was analysed by western blotting with anti-PK and anti-HA antibodies before and after auxin addition. (C) Data from Figure 2A is plotted to show the percentage of cohesin that remains on DNA upon Scc2 inactivation (after temperature shift) along the entire chromosome 4. The median cohesin level along the entire chromosome 4 (dotted line) is marked with arrowheads. (D) waplΔ (K27569) and waplΔ scc4-4 (K27570) described in Figure 3B were arrested in G1 at 25°C and released into late G1 arrest for 90 min at 37°C. Samples drawn after 90 min in late G1 were analysed by calibrated ChIP-sequencing (Scc1-PK6). Cohesin ChIP profiles showing the number of reads at each base pair away from the CDEIII element averaged over all 16 chromosomes is shown.
Figure 4 with 1 supplement
Wapl-independent release requires Smc ATPases and involves dissociation of the Smc3-Scc1 interface.
(A) smc1D1164E (K26765) and smc1D1164E scc2-45 (K26766) strains were arrested in late G1 at 25°C and subjected to temperature shift to 37°C for 20 min. Ratio of average cohesin levels before and 20 min after the temperature shift is plotted. (B) Strains expressing a covalently fused Smc3-Scc1 fusion protein, smc3-scc1 (K26994) and smc3-scc1 scc2-45 (K26993) were arrested in late G1 at 25°C and subjected to temperature shift to 37°C for 20 min. Ratio of average cohesin levels before and 20 min after the temperature shift is plotted. Also see Figure 4—figure supplement 1E. (C) Scc1 is cleaved by Separase in Anaphase. The Separase cleaved N-Terminal fragment of Scc1 (NScc1) remains stably associated with Smc3 in cells lacking Wapl mediated releasing activity. This interaction can be measured by crosslinking the two proteins in vivo using BMOE as detailed in Materials and Methods; Cys substitution of a Ser residue at 1043 in Smc3 allows the crosslinking of the 1043C with a natural Cys at position 56 in Scc1. (D) Wild type (K22156), scc2-3XmAID (28095), waplΔ (K28094) and waplΔ scc2-3XmAID (K28096) strains expressing Smc3 (S1043C)-HA3 were arrested in G2 with nocodazole. Subsequently, 5 mM auxin was added to the G2 arrested cultures and incubated for 60 min (See Figure 4—figure supplement 1F). This was followed by in vivo crosslinking and Smc3 IP as detailed in Materials and Methods. Smc3-HA3 immunoprecipitated from whole-cell extracts was analysed by western blotting detecting the HA epitope. (E) Smc3-HA3 immunoprecipitated from whole-cell extracts of strains grown (D) that were not subjected to in vivo crosslinking. The IP was analysed by western blotting against HA and MYC epitopes, the bands corresponding to full length Scc1 and the Scc1 N-terminal fragment are marked. (F) scc2-3XmAID (28095), waplΔ (K28094) and waplΔ scc2-3XmAID (K28096) strains expressing Smc3 (S1043C)-HA3 were arrested in G2 with nocodazole. The cultures were either treated with 5 mM auxin or left untreated for 60 min. This was followed by in vivo crosslinking and western blotting to detect Smc3-Scc1 crosslinks. Three independent repetitions of the experiment were quantified using the LI-COR odyssey software to measure the intensities of Smc3 and Smc3-NScc1. The ratio of Smc3-NScc1 band intensity to that of the Smc3 band is plotted. (G) Wapl-independent releasing activity causes disengagement of the Smc3-Scc1 interface.
Figure 4—figure supplement 1
Wapl-independent release requires Smc ATPases.
(A) Ratio of average cohesin levels before and after temperature shift of smc3T1185M (K27536) and smc3T1185M scc2-45 (K27537) strains arrested in late G1. (B) Data from (A) is plotted to show the percentage of cohesin that remains on DNA upon Scc2 inactivation (after temperature shift) along the entire chromosome 4. The median cohesin level along the entire chromosome 4 (dotted line) is marked with arrowheads. (C) ATPase activity of purified WT, smc1E1155Q smc3E1158Q and smc1D1164E mutant tetramer stimulated by Scc2. The rate of ATP hydrolysis (ATP molecules hydrolysed/Cohesin/minute) was measured either in the presence or absence of DNA as described in Materials and Methods. (D) Calibrated ChIP profile of smc3E1155Q (K17409) and smc3E1155Q smc1D1164E (25039). Cohesin ChIP profiles showing the number of reads at each base pair away from the CDEIII element averaged over all 16 chromosomes is shown. (E) The cultures expressing the Smc3-Scc1 fusion protein described in Figure 4B were analysed by western blotting against PK and PGK antibodies. (F) The scc2-3XmAID (K28095) and waplΔ scc2-3XmAID (28096) described in Figure 4D were analysed by western blotting with anti-mAID and anti-PGK antibodies before and after auxin addition.
Figure 5 with 2 supplements
Smc3 acetylation and DNA replication are not required to abolish Wapl-independent release.
(A) scc3K404E scc2-45 (K24709) and smc3K404 eco1Δ scc2-45 (K25947) strains arrested in G2 at 25°C and subjected to temperature shift to 37°C for 20 min. Ratio of average cohesin levels before and 20 min after the temperature shift is plotted. (B) Strains grown in (A) were analysed by western blotting against the indicated antibodies. (C) scc3K404E (K24697) and scc3K404E scc2-45 (K24738) strains containing 2C Smc1, 2C Smc3 and galactose inducible 2C Scc1NC were arrested in G2 in YEP raffinose and Scc1NC expression induced by addition of galactose. 60 min after galactose addition, glucose was added to the cultures and temperature shifted to 37°C. 0- and 20 min samples were subjected to mini-chromosomeIP (Scc1-PK6) following in vivo cohesin crosslinking with BMOE. (D) 0- and 20 min samples from (C) were subjected to Cal-ChIP-Seq. ratio of average cohesin levels before and temperature shift is plotted. Also see Figure 5—figure supplement 2B. (E) Cdc45 was depleted from wapl−AID cdc45-AID (K27169) and wapl-AID cdc45-AID scc2-45 (K27168) strains like described in Materials and Methods. Following temperature shift, 0- and 20 min samples of the cdc45 depleted strains were subjected to Cal-ChIP-Seq. ratio of average cohesin levels before and temperature shift from −100 kb to +60 KB relative to all 16 centromeres is plotted. Also see Figure 5—figure supplement 2C and D. (F) The percentage of cohesin that remains on DNA upon Scc2 inactivation (after temperature shift) along the entire chromosome four is shown. The median cohesin level along the entire chromosome 4 (dotted line) is marked with arrowheads. Data from SCC2 wild type cells arrested in lateG1 (Figure 2A) is shown in grey. Data from scc2-45 cells arrested in late G1 (Figure 2A) is shown in yellow. Data from Cdc45 depleted scc2-45 cells (Figure 5E) is shown in turquoise. Data from scc2-45 cells arrested in G2 (Figure 2B) is shown in green.
Figure 5—figure supplement 1
Neither Smc3 acetylation nor Pds5 are required to turn off Wapl-independent release.
(A) waplΔ eco1Δ (K22698) and waplΔ eco1Δ scc2-45 (K22697) strains carrying 2.3 kb circular mini-chromosome were arrested in G2 and subjected to temperature shift. Mini-chromosome dimers and monomers were separated by sucrose gradient sedimentation and gel electrophoresis and detected by Southern blotting as detailed in Materials and Methods. (B) pds5-AID (K26415) and pds5-AID scc2-45 (K26414) strains were arrested in G1 with α-factor and released into G2 arrest in the presence of auxin (IAA) and subjected to temperature shift and Cal-ChIP-Seq. ratio of average cohesin levels before and temperature shift is plotted. (C) Data from (B) is plotted to show the percentage of cohesin that remains on DNA upon Scc2 inactivation (after temperature shift) along the entire chromosome 4. The median cohesin level along the entire chromosome 4 (dotted line) is marked with arrowheads. (D) pds5-AID scc2-45 (K26414) strain was arrested in G1 with α-factor and released into G2 arrest in the presence or absence of auxin (IAA) and analysed by western blotting with anti-PK and anti-HA antibodies.
Figure 5—figure supplement 2
DNA replication is not required to turn off Wapl-independent release.
(A) scc3K404E (K24697) and scc3K404E scc2-45 (K24738) strains containing 2C Smc1 and 2C Smc3 and galactose inducible 2C Scc1NC were arrested in G2 in YEP raffinose. The culture was split into two, one half incubated at 25°C and the other at 37°C. Scc1 expression induced by addition of galactose. 60 min after galactose addition samples drawn and the pellets were subjected to mini-chromosomeIP (Scc1-PK6). (B) Data from experiment described in Figure 5D is plotted to show the percentage of cohesin that remains on DNA upon Scc2 inactivation (after temperature shift) along the entire chromosome 4. The median cohesin level along the entire chromosome 4 (dotted line) is marked with arrowheads. Strain expressing wapl-AID cdc45-AID scc2-45 (K27169) was arrested in G1 with alpha-factor and released in the presence or absence of auxin and subjected to FACS analysis along with a culture arrested in late G1 (Sic1) and G2. (C) and western blotting with indicated antibodies (D).
Figure 6
Cohesin expressed at high CDK1 levels is resistant to Wapl -independent release.
(A) CDK1 inhibition leads to cohesin cleavage: cdc28-as1(K25423) cells were arrested in G2 with nocadazole and treated with 5 μM 1NMPP1. Samples were drawn at indicated times and subjected to western blot analysis with the indicated antibodies. (B) Cohesin expressed at high CDK1 levels is resistant to Wapl-independent release: scc3K404E cdc28-as1 (K25437) and scc3K404E scc2-45 cdc28-as1 (K25440) containing 2C Smc1 and 2C Smc3 along with galactose inducible 2C Scc1NCwere arrested in G2 with nocadazole. Scc1NC expression induced by galactose addition for 60 min. Glucose and 1NMPP1 were added to the cultures for 60 min followed by temperature shift to 37°C for 20 min. Samples drawn before (0) and after temperature shift (20) were analysed by mini-chromosome IP following in vivo cohesin crosslinking with BMOE. (C) CDK1 is required to abolish Wapl-independent release: Strains described in (B) were arrested in G2. Followed by 1NMPP1 addition for 60 min. After this, galactose was added to the cultures to induce 2C Scc1NC for 60 min. Glucose was added to the cultures followed by temperature shift to 37°C for 20 min. Samples drawn before (0) and after temperature shift (20) were analysed by mini-chromosome IP following in vivo cohesin crosslinking with BMOE.
Figure 7 with 1 supplement
In the absence of all forms of cohesin release, Scc2 becomes dispensable for cohesion establishment.
(A–C) Transient inhibition of CDK1 in mitotic cells induces re-replication: (A) Growth conditions to induce re-replication: scc3K404E cdc28-as1 (K25437) containing 2C Smc1 and 2C Smc3 along with galactose inducible 2C Scc1NC was arrested in G2 with nocadazole. Scc1NC expression induced by galactose addition for 60 min. After this, glucose and 1NMPP1 were added to the cultures. After 60 min, the culture was shifted to 37°C for 20 min. Subsequently, the culture was filtered, washed with YEP medium and resuspended into YEPD medium containing either DMSO or 1NMPP1at 37°C. 90 min later, the culture was analysed by western blotting with indicated antibodies (B) and FACS (C). (D and E) in the absence of release, Scc2 is dispensable for cohesion establishment: re-replication was induced in scc3K404E cdc28-as1 (K25437) and scc3K404E scc2-45 cdc28-as1 (K25440) expressing galactose inducible 6C non-cleavable cohesin using the growth regime described in (A). Samples drawn before (D) and after (E) induction of re-replication were analysed by in vivo crosslinking and minichromosome IP. (F) Inactivation of releasing activity suppresses the cohesion defect caused by Scc2 inactivation: Sister chromatid cohesion was measured as described in Materials and Methods in pds5S81R (K27443) scc2-4 (K15028) and scc2-4 pds5S81R (K27575) strains that were arrested in G1 or S (HU) and released into G2 arrest (by Cdc20 depletion) at non-permissive temperature (35.5°C).
Figure 7—figure supplement 1
In the absence of all forms of cohesin release, Scc2 becomes dispensable for cohesion establishment.
(A) CMs and CDs in WT (K23889) and scc2-45 (K24267) 6C strains arrested in G1 with α factor at 25°C in YPD medium and released into nocodazole at 37°C. (B) FACS profiles of the strains in the experiment described in Figure 7D (1NMPP1) and E (DMSO). (C) Two independent repetitions of the experiment described in Figure 7D and E. (D) CDs formed after re-replication are dependent on cohesin circularisation with BMOE: re-replication was induced in scc3K404E cdc28-as1 (K25437) and scc3K404E scc2-45 cdc28-as1 (K25440) expressing galactose inducible 6C non-cleavable cohesin using the growth regime described in Figure 7A. Samples that were crosslinled with BMOE were analysed along with those that were not crosslinked with BMOE by mini-chromosome IP. (E) Wild type strain (K699) was released from a G1 arrest into nocadazole or HU containing medium for indicated times and subjected to calibrated whole genome deep sequencing as described in Materials and Methods. The reads from 45 min HU, 90 min HU and G2 were normalised with the reads from G1 arrested culture (un-replicated, 1 copy of the genome). A portion of chromosome V is shown with the positions of ARS elements and URA3 gene marked. (F) Sister chromatid cohesion was measured in scc2-4 pds5S81R strain (K27575) that was arrested in S (HU) and released into G2 arrest (by Cdc20 depletion) at either permissive (blue curve) or non-permissive temperature (red curve).
Tables
Key resources table
| Reagent type (Species) or resource | Designation | Source Or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ All following strains are based on this background | This study | K699 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa scc2-4 SCC1-PK9 TETR-GFP::LEU2 TET Os::URA3 trp1:MET3p-CDC20::TRP1 | This study | K15028 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae Mata trp1:smc3(E1155Q)-PK6::TRP1 | This study | K17409 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae Mata SCC1-PK9::KAN MX rad61Δ::HGH MX leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4 (single copy) | This study | K20891 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae Mat a MATa SCC1-PK9::KanMX rad61Δ::HGH MX smc3(R1008I)::HIS3 scc2-45::NAT MX (L545P D575G) leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4(single copy) | This study | K22294 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae Mat a MATa SCC1-PK9::KanMX rad61Δ::HGH MX smc3(R1008I)::HIS3 leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4(single copy) | This study | K22296 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa eco1Δ::NatMX4 rad61Δ::HGH MX scc2-45::NAT MX (L545P D575G) Smc3(E199A/R1008I)::HIS37.5 kb minichromosome (TRP1 ARS1 CEN1-6KB) | This study | K22297 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa eco1Δ::NatMX4 rad61Δ::HGH MX Smc3(E199A/R1008I)::HIS3 7.5 kb minichromosome (TRP1 ARS1 CEN1-6KB) | This study | K22298 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa scc1(A547C)-PK6::KanMX smc3(E570C,S1043C)::ADE2 smc1(G22C,K639C)::NatMX2.3 kb Trp1-ARS1-Cen4 plasmid | Srinivasan et al. (2018) | K23889 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa scc1(A547C)-PK6::KanMX smc3(E570C,S1043C)::ADE2 smc1(G22C,K639C)::NatMX leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4 (single copy) 2.3 kb Trp1-ARS1-Cen4 plasmid | Srinivasan et al. (2018) | K23972 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa Scc1(A547C)-pk6::KanMX Smc3(E570C,S1043C)::ADE2 Smc1(G22c,K639C)::NatMXscc2-45::natMX (L545P D575G) 2.3 kb Trp1-ARS1-Cen4 plasmid | Srinivasan et al. (2018) | K24267 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa Smc1(G22C,K639C)::NatMX4 Smc3(E570C,S1043C)::ADE2 leu2::Gal-Scc1(R180E,R268D, A547C)-PK6::LEU2 SCC3 (K404E)-HA3::HIS 2.3 kb Trp1-ARS1-Cen4 plasmid | This study | K24697 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa Scc1(A547C)-pk6::KanMX Smc3(E570C,S1043C)::ADE2 Smc1(G22c,K639C)::NatMX scc2-45::natMX (L545P D575G) scc3 (K404E)-HA3::HIS 2.3 kb Trp1-ARS1-Cen4 plasmid | This study | K24709 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa Smc1(G22C,K639C)::NatMX4 Smc3(E570C,S1043C)::ADE2 leu2::Gal-Scc1(R180E,R268D, A547C)-PK6::LEU2 scc3 (K404E)-HA3::HIS scc2-45::natMX (L545P D575G) TRP1-ARS1-CEN4 2.3 KB plasmid | This study | K24738 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa trp1::smc3(E1155Q)- PK6::TRP1 smc1(D1164E) | This study | K25039 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa Scc1(A547C)-pk6::KanMX Smc3(E570C,S1043C)::ADE2 Smc1(G22c,K639C)::NatMX scc2-45::natMX (L545P D575G) 2.3 kb Trp1-ARS1-Cen4 plasmid | This study | K25238 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa scc1(A547C)-PK6::KanMX pds5 (s81R)::HIS3 smc3(E570C,S1043C)::ADE2 smc1(G22C,K639C)::NatMX leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4 (single copy) 2.3 kb Trp1-ARS1-Cen4 plasmid | This study | K25311 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa scc1(A547C)-PK6::KanMX pds5 (s81R)::HIS3 smc3(E570C,S1043C)::ADE2 smc1(G22C,K639C)::NatMX scc2-45::natMX (L545P D575G) leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4 (single copy) 2.3 kb Trp1-ARS1-Cen4 plasmid | This study | K25312 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa scc1(A547C)-PK6::KanMX scc3 (K404e)-HA3::HIS3 smc3(E570C,S1043C)::ADE2 smc1(G22C,K639C)::NatMX leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4 (single copy) 2.3 kb Trp1-ARS1-Cen4 plasmid | This study | K25313 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa scc1(A547C)-PK6::KanMX scc3 (K404e)-HA3::HIS3 smc3(E570C,S1043C)::ADE2 smc1(G22C,K639C)::NatMX scc2-45::natMX (L545P D575G) leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4 (single copy) 2.3 kb Trp1-ARS1-Cen4 plasmid | This study | K25316 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa scc1(A547C)-PK6::KanMX smc3(E570C,S1043C)::ADE2 smc1(G22C,K639C)::NatMX scc2-45::natMX (L545P D575G) cdc28-as1 | This study | K25423 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa smc3(E570C,S1043C)::ADE2 smc1(G22C,K639C)::NatMX leu2::Gal-Scc1(R180E,R268D, A547C)-PK6::LEU2 scc3 (K404e)-HA3::HIS3 cdc28-as1 2.3 kb Trp1-ARS1-Cen4 plasmid | This study | K25437 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa smc3(E570C,S1043C)::ADE2 smc1(G22C,K639C)::NatMX leu2::Gal-Scc1(R180E,R268D, A547C)-PK6::LEU2 scc2-45::natMX (L545P D575G) scc3 (K404e)-HA3::HIS3 cdc28-as1 2.3 kb Trp1-ARS1-Cen4 plasmid | This study | K25440 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa smc3(E570C,S1043C)::ADE2 smc1(G22C,K639C)::NatMX scc1(A547C)-PK6::KanMX scc2-45::natMX (L545P D575G) eco1Δ::HGH MX scc3 (K404E)-HA3::HIS TRP1-ARS1-CEN4 2.3 KB plasmid | This study | K25947 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa ura::ADH1 promoter-OsTIR1-9myc::URA3 PDS5-Pk3-aid::KanMX4scc2-45::NatMX scc3 (K404E)::HA3::HIS leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4::Leu2 (single copy) | This study | K26414 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa ura::ADH1 promoter-OsTIR1-9myc::URA3 PDS5-Pk3-aid::KanMX4 scc3 (K404E)::HA3::HIS leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4::Leu2 (single copy) | This study | K26415 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa smc1(D1164E)::HIS3MX SCC1-PK9::KanMX leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4::Leu2 (single copy) | This study | K26765 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa smc1(D1164E)::HIS3MX SCC1-PK9::KanMX scc2-45::natMX (L545P D575G) leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/ Gal1p-Gal4::Leu2 (single copy) | This study | K26766 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa ura::ADH1 promoter-OsTIR1-9myc::URA3 scc2-3XmAID::KANMX6 Scc1-PK9::KanMX rad61Δ::HGH MX leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4::Leu2 (single copy) | This study | K26831 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa scc2-45::natMX (L545P D575G) leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4::Leu2 (single copy) ura3::Scc1P-Smc3(E570C)-TEV3-Scc1(A547C)-PK9::KanMX::URA3 (single integrant,fusion linker: (GGGGS)x8 + TEV3) | This study | K26993 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa scc2-45::natMX (L545P D575G) leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4::Leu2 (single copy) Smc1(G22C, K639C)::NAT MX ura3::Scc1P-Smc3(E570C)-TEV3-Scc1(A547C)-PK9::KanMX::URA3 (single integrant,fusion linker: (GGGGS)x8 + TEV3) | This study | K26994 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa bar1::hisG ura3-1::GAL-OsTIR1-9Myc::URA3 cdc45-AID (CDC45-5xGA-IAA17)::kanMX rad61-3slAA::natNT2 SCC1-PK6::TRP1 scc2-45::natMX (L545P D575G) | This study | K27168 | Materials and methods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa bar1::hisG ura3-1::GAL-OsTIR1-9Myc::URA3 cdc45-AID (CDC45-5xGA-IAA17)::kanMX rad61-3slAA::natNT2 SCC1-PK6::TRP1 | This study | K27169 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa TET R-GFP::LEU2 TETOs::URA3 pds5 S81R-HA6::HIS (NO HA tag) trp1:MET3p-CDC20::TRP1 | This study | K27443 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa SCC1-PK9::KanMX smc3(T1185M)-HIS3M × 6 scc2-45::natMX (L545P D575G) leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4::Leu2 (single copy) | This study | K27536 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa SCC1-PK9::KanMX smc3(T1185M)-HIS3M × 6 leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4::Leu2 (single copy) | This study | K27537 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa SCC1-PK9::KanMX rad61Δ::hghMX leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4::Leu2 (single copy) | This study | K27569 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa SCC1-PK9::KanMX rad61Δ::hghMX scc4-2:NAT leu2::Gal1p-Sic1(9 m)/His3p-Gal1/His3p-Gal2/Gal1p-Gal4::Leu2 (single copy) | This study | K27570 | Materials andmethods subsection experimental models |
| Strain, strain background (S. cerevisiae) | S. cerevisiae MATa scc2-4 TET R-GFP::LEU2 TETOs::URA3 pds5 S81R-HA6::HIS (NO HA tag) trp1:MET3p-CDC20::TRP1 | This study | K27575 | Materials andmethods subsection experimental models |
| Strain, strain background (Spodoptera frugiperda) | Sf9 insect cells | ThermoFisher | Cat# 11496015 | |
| Antibody | Mouse monoclonal Anti-V5 | BioRad | Cat# MCA1360 | (1:1000) |
| Antibody | Mouse monoclonal Anti Smc3 Acetyl | Beckouët et al. (2010) | N/A | (1:1000) |
| Antibody | Rabbit polyclonal Anti Clb2 y-180 | SantaCruz | Sc-9071 | (1:1000) |
| Antibody | Rabbit polyclonal Anti Sic1 FL-284 | SantaCruz | Sc-50441 | (1:1000) |
| Antibody | Mouse monoclonal Anti PGK1 | ThermoFisher Scientific | Cat#459250 | (1:5000) |
| Antibody | Rabbit polyclonal Anti-mini-AID-tag mAb | Kanemaki MT/MBL International | M214-3 | (1:1000) |
| Chemical compound, drug | Acid-washed glass beads | Sigma | Cat# G8722 | |
| Chemical compound, drug | ATP α-32P | Hartmann Analytic | Cat# SRP-203 | |
| Chemical compound, drug | Bismaleimidoethane (BMOE) | ThermoFisher | Cat# 22323 | (5 mM) |
| Chemical compound, drug | Complete EDTA free protease inhibitor cocktail | Roche | Cat# 4693132001 | (1:50 ml) |
| Chemical compound, drug | Dithiothreitol | Fluka | Cat# BP172 | (5 mM) |
| Chemical compound, drug | DMSO | Sigma | Cat# D8418 | |
| Chemical compound, drug | Immobilon Western ECL | Millipore | Cat# WBLKS0500 | |
| Chemical compound, drug | Trisodium citrate | Sigma | Cat# W302600 | |
| Chemical compound, drug | RNase A | Roche | Cat# 10109169001 | |
| Chemical compound, drug | Nocodazole | Sigma | Cat# M1404 | |
| Chemical compound, drug | 1NMPP1 | MerkMillipore | 529581 | |
| Chemical compound, drug | Indole-3-acetic acid (auxin) | Sigma | Cat# I3750-5G-A | |
| Chemical compound, drug | PMSF | Sigma | Cat# 329-98-6 | |
| Chemical compound, drug | Potassium chloride | Sigma | Cat# P5405 | |
| Chemical compound, drug | Proteinase K | Roche | Cat# 03115836001 | |
| Chemical compound, drug | Sodium sulfite | Sigma | Cat# 71988 | |
| Peptide, recombinant protein | α-factor peptide | CRUK Peptide Synthesis Service | ||
| Biological sample (Saccharomyces cerevisiae) | Saccharomyces cerevisiae cohesin | This study | Materials and methods subsection Tetramer and Scc2 purification | |
| Biological sample (Saccharomyces cerevisiae) | Saccharomyces cerevisiae Scc2 | This study | Materials andmethods subsection Tetramer and Scc2 purification | |
| Commercial assay or kit | AxyPrep Mag PCR Clean up Kit | Appleton Woods Ltd. | Cat# AX402 | |
| Commercial assay or kit | ChIP Clean and Concentrator Kit | Zymo Research | Cat# D5205 | |
| Commercial assay or kit | E-Gel SizeSelect II Agarose Gels, 2% | ThermoFisher | Cat# G661012 | |
| Commercial assay or kit | EnzChek phosphate assay kit | Invitrogen | Cat# E6646 | |
| Commercial assay or kit | HiTrap TALON column | GE Healthcare | Cat# 28-9537-67 | |
| Commercial assay or kit | Library Quantification Kit Ion Torrent Platforms | KAPA Biosystems | Cat# KR0407 | |
| Commercial assay or kit | Microcon YM-100 columns | Sigma | Cat# Z648094 | |
| Commercial assay or kit | NEBNext Fast DNA library prep set for Ion Torrent | NEB | Cat# E6270L | |
| Commercial assay or kit | NuPAGE 3–8% Tris-Acetate Protein Gels, 1.5 mm, 10-well | ThermoFisher | Cat# EA0378BOX | |
| Commercial assay or kit | NuPAGE 4–12% Bis-Tris Protein Gels, 1.0 mm, 10-well | ThermoFisher | Cat# NP0321BOX | |
| Commercial assay or kit | Prime-it II Random Primer Labelling Kit | Agilent | Cat# 300385 | |
| Commercial assay or kit | Protein G dynabeads | ThermoFisher | Cat# 10003D | |
| Commercial assay or kit | Slide-a-lyzer dialysis units (3.5 kDa) | ThermoFisher | Cat# 66330 | |
| Commercial assay or kit | StrepTrap HP column | GE Healthcare | Cat# 28-9075-48 | |
| Commercial assay or kit | Superdex 200 16/60 GL | GE Healthcare | Cat# 17-1069-01 | |
| Commercial assay or kit | Superose 6 10/300 GL | GE Healthcare | Cat# 17517201 | |
| Commercial assay or kit | TALON Superflow metal affinity resin | Clontech | Cat# 635670 | |
| Software, algorithm | Galaxy platform | Giardine et al. (2005) | https://usegalaxy.org | |
| Software, algorithm | FastQC | Galaxy tool version 1.0.0 | https://usegalaxy.org | |
| Software, algorithm | Trim sequences | Galaxy tool version 1.0.0 | https://usegalaxy.org | |
| Software, algorithm | Filter FASTQ | Galaxy tool version 1.0.0 | https://usegalaxy.org | |
| Software, algorithm | Bowtie2 | Langmead and Salzberg (2012) Galaxy tool version 0.2 | https://usegalaxy.org | |
| Software, algorithm | Bam to BigWig | Galaxy tool version 0.1.0 | https://usegalaxy.org | |
| Software, algorithm | Samtools | Li et al. (2009) | http://samtools.sourceforge.net/ | |
| Software, algorithm | IGB browser | Nicol et al. (2009) | http://bioviz.org/igb/ | |
| Software, algorithm | Filter SAM or BAM | Li et al. (2009) Galaxy tool version 1.1.0 | https://usegalaxy.org | |
| Software, algorithm | chr_position.py | This study | https://github.com/naomipetela/nasmythlab-ngs | |
| Software, algorithm | filter.py | This study | https://github.com/naomipetela/nasmythlab-ngs | |
| Software, algorithm | bcftools call | Li et al. (2009) |
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Scc2 counteracts a Wapl-independent mechanism that releases cohesin from chromosomes during G1
eLife 8:e44736.
https://doi.org/10.7554/eLife.44736
