Cohesion is established during DNA replication utilising chromosome associated cohesin rings as well as those loaded de novo onto nascent DNAs
- Department of Biochemistry, University of Oxford, United Kingdom
- Department of Molecular and Cellular Biology, Harvard University, United States
Figures
Figure 1
Possible mechanisms of cohesion establishment.
(A) Cohesin complexes previously associated with un-replicated DNA converted into cohesive ones behind the replication fork (Cohesin conversion) or cohesion built by de novo loading of cohesin …
Figure 2 with 1 supplement
Chromosomal cohesin is converted into cohesive structures in the absence of Scc2 activity.
(A) A schematic depiction of the cohesin conversion assay, see the results and Materials and methods sections for detailed description of the assay. Green rings denote endogenous cohesin and red …
Figure 2—figure supplement 1
Non cleavable cohesin expressed in the G2 phase survives mitosis and remains stably associated with the chromosomes in the subsequent G1 phase.
(A) 6C non cleavable cohesin was expressed for 45 min in Wild type (K24697) strain arrested in G2 phase. The culture was released from the G2 arrest and arrested in the subsequent the G1 phase, a …
Figure 3 with 1 supplement
Tof1/Csm3, Ctf4 and Chl1 are essential for cohesin conversion.
(A) Exponentially growing wild type (K23889), mrc1Δ (K28092), chl1Δ (K28082), ctf4Δ (K28084), csm3Δ (K28108), tof1Δ (K28091) and ctf18Δ (K28115) expressing endogenous 6C cohesin were synchronised in …
Figure 3—figure supplement 1
FACS profiles of the cultures described in Figure 3B.
Figure 4 with 2 supplements
Chromosome associated cohesin is evicted during S phase in the absence of the conversion pathway.
(A) Wild type (K24697), scc2-45 (K24738), chl1Δ (K28175) and chlΔ scc2-45 (K28061) strains that contain genes coding for 6C non cleavable cohesin (2C SMC1 2C SMC3 and GALp-2C SCC1NC) were arrested …
Figure 4—figure supplement 1
Chromosome associated cohesin is evicted during S phase in the absence of the conversion pathway.
(A) The haploid progeny from crossing a chl1Δ and scc2-45 strain are shown, the genotype of selected haploids is shown. (B) The cultures that described in Figure 4A, after undergoing replication at …
Figure 4—figure supplement 2
The occupancy of Scc1NC along chromosome IV in the experiment described in Figure 4B.
Figure 5 with 1 supplement
Cohesion can also be established by CTF18-RFC and Scc2 dependent de novo loading of nucleoplasmic cohesin.
(A) Wild type (K24697), chl1Δ (K28175), ctf4Δ (K28275), csm3Δ (K28282) and tof1Δ (K28280) strains that contain genes coding for 6C non cleavable cohesin (2C SMC1 2C SMC3 and GALp-2C SCC1NC) were …
Figure 5—figure supplement 1
Cohesion can also be established by CTF18-RFC and Scc2 dependent de novo loading of nucleoplasmic cohesin.
(A) chl1Δ (K28175) and chl1Δ scc2-45 (K28061) cultures from the final step of the experiment described in Figure 5B were analysed by western botting against the indicated antibodies and in (B), the …
Figure 6 with 1 supplement
Augmentation of either cohesion establishment pathway suppresses the cohesion defects caused by abrogation of the other.
(A) Asynchronous cultures of wild-type (K10003), ctf4Δ (K11692), ctf8Δ (K10349) that carry either an empty 2μ vector or a 2μ vector containing the CHL1 gene were fixed and stained for DNA and with …
Figure 6—figure supplement 1
The fitness of wild type, ctf18Δ and ctf4Δ strains relative to the wild-type ancestor.
Error bars represent standard deviation, the p values from unequal variances t-tests are also shown.
Figure 7
Two parallel pathways for cohesion establishment at the replication fork: chromosome associated cohesin is converted into cohesive structures during S phase.
This process does not require Scc2 (except to load cohesin in the first place), but is strictly dependent on Tof1, Csm3, Chl1, and Ctf4 (the TCCC pathway). A second pathway that operates in parallel …
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (S. cerevisiae) | NCBITaxon:4932 | This paper | Yeast strains | Supplementary file 1 |
| Antibody | Mouse monoclonal Anti-V5 | BioRad | Cat# MCA1360 | (1:1000) |
| Antibody | Mouse monoclonal Anti PGK1 | ThermoFisher Scientific | Cat#459250 | (1:5000) |
| Chemical compound | Acid-washed glass beads | Sigma | Cat# G8722 | N/A |
| Chemical compound | ATP α-32P | Hartmann Analytic | Cat# SRP-203 | N/A |
| Chemical compound | Bismaleimidoethane (BMOE) | ThermoFisher | Cat# 22323 | (5 mM) |
| Chemical compound | Complete EDTA free protease inhibitor cocktail | Roche | Cat# 4693132001 | (1:50 ml) |
| Chemical compound | Dithiothreitol | Fluka | Cat# BP172 | (5 mM) |
| Chemical compound | DMSO | Sigma | Cat# D8418 | N/A |
| Chemical compound | Immobilon Western ECL | Millipore | Cat# WBLKS0500 | N/A |
| Chemical compound | Trisodium citrate | Sigma | Cat# W302600 | N/A |
| Chemical compound | RNase A | Roche | Cat# 10109169001 | N/A |
| Chemical compound | Nocodazole | Sigma | Cat# M1404 | N/A |
| Chemical compound | PMSF | Sigma | Cat# 329-98-6 | N/A |
| Chemical compound | Potassium chloride | Sigma | Cat# P5405 | N/A |
| Chemical compound | Proteinase K | Roche | Cat# 03115836001 | N/A |
| Chemical compound | Sodium sulfite | Sigma | Cat# 71988 | N/A |
| Biological Sample | α-factor peptide | CRUK Peptide Synthesis Service | N/A | N/A |
| Commercial Assay or Kit | ChIP Clean and Concentrator Kit | Zymo Research | Cat# D5205 | N/A |
| Commercial Assay or Kit | E-Gel SizeSelect II Agarose Gels, 2% | ThermoFisher | Cat# G661012 | N/A |
| Commercial Assay or Kit | Library Quantification Kit Ion Torrent Platforms | KAPA Biosystems | Cat# 28-9537-67 | N/A |
| Commercial Assay or Kit | NEBNext Fast DNA library prep set for Ion Torrent | NEB | Cat# Z648094 | N/A |
| Commercial Assay or Kit | NuPAGE 3–8% Tris-Acetate Protein Gels, 1.5 mm, 10-well | ThermoFisher | Cat# E6270L | N/A |
| Commercial Assay or Kit | Prime-it II Random Primer Labelling Kit | Agilent | Cat# NP0321BOX | N/A |
| Commercial Assay or Kit | Protein G dynabeads | ThermoFisher | Cat# 300385 | N/A |
| Software, algorithm | Galaxy platform | Giardine et al., 2005 | ||
| Software, algorithm | FastQC | Galaxy tool version 1.0.0 | https://usegalaxy.org | N/A |
| Software, algorithm | Trim sequences | Galaxy tool version 1.0.0 | https://usegalaxy.org | N/A |
| Software, algorithm | Filter FASTQ | Galaxy tool version 1.0.0 | https://usegalaxy.org | N/A |
| Software, algorithm | Bowtie2 | Langmead and Salzberg, 2012 Galaxy tool version 0.2 | https://usegalaxy.org | N/A |
| Software, algorithm | Bam to BigWig | Galaxy tool version 0.1.0 | https://usegalaxy.org | N/A |
| Software, algorithm | Samtools | Li et al., 2009 | https://usegalaxy.org | N/A |
| Software, algorithm | IGB browser | Nicol et al., 2009 | http://samtools.sourceforge.net/ | N/A |
| Software, algorithm | Filter SAM or BAM | Li et al., 2009 Galaxy tool version 1.1.0 | http://bioviz.org/igb/ | N/A |
| Software, algorithm | chr_position.py | This study | https://usegalaxy.org | N/A |
| Software, algorithm | filter.py | This study; Petela, 2019 | https://github.com/naomipetela/nasmythlab-ngs | N/A |
| Software, algorithm | bcftools call | Li et al., 2009 | https://github.com/naomipetela/nasmythlab-ngs | N/A |
Additional files
-
Source data 1
Source data for the Relative fitness assay.
- https://cdn.elifesciences.org/articles/56611/elife-56611-data1-v1.xlsx
-
Supplementary file 1
Strain list.
- https://cdn.elifesciences.org/articles/56611/elife-56611-supp1-v1.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/56611/elife-56611-transrepform-v1.docx
