1. Volume 23, Issue 6, Pages 787-799 (September 2006)
Establishment of Sister Chromatid Cohesion at the S. cerevisiae Replication Fork 
Armelle Lengronne, John McIntyre, Yuki Katou, Yutaka Kanoh, Karl-Peter Hopfner, Katsuhiko Shirahige, Frank Uhlmann 
Molecular Cell 
Volume 23, Issue 6, Pages (September 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Cohesin Loading in G2/M
(A) Scc2-dependent cohesin loading in G2/M to sites used by G1-loaded cohesin. Strains Y2636 (MATa GAL-SCC1-HA3) and Y2637 (MATa scc2-4 GAL-SCC1-HA3) were arrested in G2/M by nocodazole treatment. scc2-4 was inactivated for 1 hr at 37°C, and GAL-SCC1-HA was induced by galactose addition for 1 hr. After formaldehyde crosslinking and DNA fragmentation, Scc1 was precipitated, and enrichment of DNA fragments in the immunoprecipitate relative to a whole genome sample is shown along the right arm of chromosome VI. Localization of endogenous Scc1-HA6 (K8869) and Scc2-HA6 (Y1686) in nocodazole-arrested cells is shown for comparison. Each bar represents the average of 16 oligonucleotide probes within adjacent 300 bp windows. The y axis scale is log2. Gray signals represent significant binding as described (Katou et al., 2003). Blue bars above and below the midline are genes transcribed from left to right and opposite, respectively. The centromere is depicted in dark red, origins of replication in red, tRNA genes in yellow, and Ty elements in green.
(B) G2/M-loaded cohesin does not establish sister chromatid cohesion. MET-CDC20 scc1-73 GAL-SCC1-HA3 TetOs::URA3 TetR-GFP cells (Y1313) were arrested in G2/M by Cdc20 depletion. Wild-type Scc1 was induced before arrest (+Gal-Scc1-HA) or for 1 hr after cells reached the arrest (+Gal-Scc1-HA in G2/M), or was not induced (−Gal-Scc1-HA). Cells were shifted to 37°C (time, 0 min) to inactivate scc1-73, and separation of the GFP-marked URA3 locus was monitored. Stable arrest in G2/M throughout the experiment was verified by FACS analysis of DNA content, and chromosome binding of GAL1-expressed cohesin was confirmed by immunostaining of chromosome spreads (data not shown).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Cohesion Establishment during S Phase by Preloaded Cohesin
(A) Scc2-dependent cohesin loading is not required during S phase. MATa scc2-4 (K5828) and MATa eco1-1 (Y1013) strains were released after α factor synchronization into HU arrest in early S phase at 23°C and 35.5°C. Cell viability was determined in both cultures every 20 min by plating of an aliquot at 23°C. After 1 hr in HU, half of the culture at 23°C was shifted to 35.5°C for 40 min, and all three cultures were then released into nocodazole arrest. One hundred percent viability reflects the average number of viable cells throughout the timecourse at 23°C.
(B) No Scc2-independent cohesin loading during S phase. SCC1-Pk9 GAL-SCC1-HA3 (Y2278) and scc2-4 SCC1-Pk9 GAL-SCC1-HA3 (Y2279) cells were synchronized in G1 using α factor. Cells were released into HU arrest at 25°C and shifted to 37°C to inactivate scc2-4. After 1 hr, GAL-SCC1-HA3 was induced by galactose (Gal) addition for 1.5 hr, and cells were then released into nocodazole (Noc) containing media at 37°C. The pattern of Scc1-HA is shown after 1.5 hr in nocodazole on the chromosome VI left arm. The green dashed lines flank an Scc2 binding site (Lengronne et al., 2004). FACS analysis of DNA contents throughout the experiment is shown on the right.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Proteins Involved in Sister Chromatid Cohesion Localize to the Replication Fork
(A) Eco1, Ctf4, and Ctf18 localize to replication forks arrested with HU. MATa GPD-TK CTF18-HA6 (Y1491), MATa ECO1-Flag3 (Y1621), and MATa CTF4-HA6 (Y1451) strains were synchronized in G1 and released into HU arrest for 1 hr. Strain Y1491 was released in the presence of 400 μg/ml of bromodeoxyuridine (BrdU). ChIP was performed, and localization of BrdU or the respective proteins is shown along chromosome VI.
(B) Ctf4 travels from replication origins bidirectionally along chromosomes during S phase. MATa CTF4-Pk9 (Y2374) cells were synchronized in G1 by α factor treatment and released into S phase at 16°C to slow down cell cycle progression. An overlay of Ctf4 distribution along chromosome VI at 40, 50, and 60 min after release is shown. The pattern at each individual time point is documented in Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Dependency of Ctf18 Localization on Ctf4 and of PCNA on Ctf18
(A) CTF4 deletion affects Ctf18 binding, but not its localization. MATa CTF18-HA6 (Y1235) and MATa ctf4Δ CTF18-HA6 (Y1613) cells were synchronized in G1 by α factor treatment and released into HU arrest. Ctf18-HA6 localization along chromosome VI is shown in both strains.
(B) MATa CTF18-myc9 (Y1198), MATa ctf4Δ CTF18-myc9 (Y1439), MATa CTF4-myc9 (Y1421), and MATa CTF4-myc9 ctf18Δ (Y1440) cells were synchronized in G1 by α factor treatment and released into either HU or nocodazole-containing media. Cell extracts (WCE) were separated into supernatant (SU) and chromatin (CH) fractions, and myc-tagged proteins were detected by immunoblotting. Hmo1 served as a chromosomal loading control (Lu et al., 1996).
(C) PCNA binding at replication forks is reduced in the absence of Ctf18. MATa Flag3-PCNA (Y1481) and MATa ctf18Δ Flag3-PCNA (Y1615) cells were synchronized in G1 by α factor treatment and released into HU arrest. Cells were processed for ChIP, and chromatin immunoprecipitates were analyzed by quantitative PCR and hybridization to the chromosome VI microarray. P197 and P259 are primer pairs covering regions next to the early replication origin ARS607 (active in HU arrest) and ARS609 (inactive in HU), respectively.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Cohesin Distribution after S Phase in Cohesion Establishment Mutant Cells
(A and B) Cohesin levels and localization on chromatin are unaffected in cohesion establishment mutants. MATa SCC1-HA6 (K8869), MATa ctf4Δ SCC1-HA6 (Y1487) MATa ctf18Δ SCC1-HA6 (Y1486), and MATa eco1-1 SCC1-HA6 (Y1311) cells were synchronised in G1 with α factor and released into nocodazole-containing medium to arrest cells in G2/M. Localization by ChIP of Scc1 is shown along the chromosome VI left arm. Levels of Scc1 on chromosomes were analyzed by cell fractionation. Orc6 and Hmo1 serve as chromosomal loading controls.
(C) Cohesin distribution is undisturbed following replication of eco1-1 cells. K8869 and Y1311 cells were synchronized in G1 with α factor, released into HU, shifted to 37°C to inactivate eco1-1 for 1 hr, and then released to progress through synchronous S phase.
(D) Maintenance of the cohesin pattern during replication of ctf18-td cells does not require Scc2. MATa scc2-4 GAL-HA-UBR1 ctf18-td-myc SCC1-Pk9 (Y2619) cells were synchronized in G1 and released into an HU block. Ctf18 and Scc2 were inactivated by temperature shift before release into G2/M arrest by nocodazole. Scc1 localization along the chromosome VI left arm is shown. As a control, MATa scc2-4 SCC1-HA6 (Y1963) cells were analyzed in the same way.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Cohesin's Arginine Fingers Are Important for Binding to DNA, but Not for Cohesion Establishment
(A) Delayed chromosome binding of arginine finger mutant “Smc1R/Smc3R” cohesin. Cultures of strains Y2329 (MATa Scc1-Pk3) and Y2330 (as Y2329, but smc1R58A smc3R58A) were released after synchronization with α factor into nocodazole-containing medium to arrest cells in G2/M. Cell extracts were fractionated into supernatant and chromatin fractions. Cell cycle progression was followed by FACS analysis of DNA content (data not shown).
(B) The cohesion defect due to arginine finger mutation is rescued by halting cell with HU before S phase. Strains Y2738 (MATa GAL-CDC20 TetR-GFP TetOs::URA3 Scc3-Pk3) and Y2739 (as Y2738 but smc1R58A smc3R58A) were released from synchronization with α factor into a G2/M block by Cdc20 depletion. Half of the culture was halted for 2 hr in an HU-imposed arrest before being allowed to complete S phase (time points of this culture are after release from HU). The GFP-marked URA3 locus was visualized to assess sister chromatid cohesion. Photographs show cells at 210 min.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Two Models for Cohesion Establishment during Replication Fork Passage
(A) The replication fork slides through the cohesin ring. A model of the replication fork is shown, very roughly to scale, based on ideas about the prokaryotic replisome (Johnson and O'Donnell, 2005). Additional proteins are thought to associate with the eukaryotic replication fork, including Mcm10, Cdc45, Sld2, Sld3, Dpb11, Tof1, Mrc1, and Csm3. Each of the known fork components should fit through cohesin; however, their three-dimensional arrangement and thus the overall dimension of the replisome are not known.
(B) Alternatively, cohesin loses its topological contact with DNA during fork passage. Cohesion establishment factors maintain the cohesin ring close to the fork and enable reassociation of cohesin with the replication products after fork passage.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions