Cohesin's concatenation of sister DNAs maintains their intertwining

Abstract

The contribution of DNA catenation to sister chromatid cohesion is unclear partly because it has never been observed directly within mitotic chromosomes. Differential sedimentation-velocity and gel electrophoresis reveal that sisters of 26 kb circular minichromosomes are held together by catenation as well as by cohesin. The finding that chemical crosslinking of cohesin's three subunit interfaces entraps sister DNAs of circular but not linear minichromosomes implies that cohesin functions using a topological principle. Importantly, cohesin holds both catenated and uncatenated DNAs together in this manner. In the vicinity of centromeres, catenanes are resolved by spindle forces, but linkages mediated directly by cohesin resist these forces even after complete decatenation. Crucially, persistence of catenation after S phase depends on cohesin. We conclude that by retarding Topo II-driven decatenation, cohesin mediates sister chromatid cohesion by an indirect mechanism as well as one involving entrapment of sister DNAs inside its tripartite ring.

Graphical abstract

Figure 1

Detection of Sister Chromatin Intertwining in Mitotic Cells (A) Separation and detection by differential sedimentation and Southern blot, respectively, of monomers (dashed orange boxes) and dimers (dashed purple boxes) of large circular minichromosomes extracted from wild-type cells (K16150) grown at 25°C. Following heat denaturation in 1% SDS, the minichromosome population is resolved into six different DNA species. (B) TOP2 protein inactivation by growth of top2-4 strain (K17890) at 37°C converts the entire minichromosome population into catenated DNAs. (C) Identification by treatment with restriction enzymes (StuI and ApaI), nicking enzyme (Nt.BbvCI), and recombinant topoisomerase II protein (Topo II) of the six individual DNA species (from bottom upwards): supercoiled circle, linear monomer, supercoiled-supercoiled catenanes, nicked circle, nicked-supercoiled catenanes, and nicked-nicked catenanes. (D) Documenting the formation of DNA catenanes during a cell cycle. Wild-type (K16150) and top2-4 (K17890) cells arrested with α factor pheromone were released at 37°C into rich media containing nocodazole. Time points were collected every 20 min for genomic DNA preparation and FACS.

Figure 2

Chromosome Arms as well as Centromeres Generate Physical Cohesion (A) Asynchronous K18066 cultures having the Z. rouxii recombinase under the control of the Gal1 promoter (Gal1P-Rec) and a 26 kb circular minichromosome containing RS-Cen3-RS (whose centromere can be uncoupled upon recombinase expression) were grown at 30°C in YEPraff; glucose (Cen3 present) or galactose (Cen3 deleted) were added to 2% final concentration for 3 hr. Cells were shifted into YPD for 1.5 hr, followed by nocodazole arrest for 1 hr 40 min. (B) K18066 cultures having the 26 kb circular minichromosome bearing the RS-Cen3-RS were arrested at 30°C with the α factor pheromone in YEPraff and released for 1 hr in nocodazole. Glucose or galactose were added to 2% final concentration and incubated for an additional 3 hr, maintaining the nocodazole arrest.

Figure 3

Measuring Sister Chromatid Cohesion of Linear Minichromosomes (A) Schematic representation of the 42 kb linear minichromosome (modified from Murray et al., 1986, with permission from Elsevier). (B) Differential sedimentation and Southern blot resolution of monomers (dashed orange boxes) and dimers (dashed purple boxes) of linear minichromosomes extracted from wild-type cells (K18251) arrested in metaphase. (C) Linear minichromosome profiles obtained from G1-arrested cells (K18251) contain exclusively monomeric DNA species. (D) SCC1's inactivation by cellular growth of scc1-73 strain (K18249) for 2 hr at 37°C in the presence of nocodazole causes the loss of linear minichromosome dimers.

Figure 4

Chemical Circularization of Cohesin Entraps Circular but Not Linear Sister Minichromosome DNAs (A–C) Dimer fractions held together by modified cohesin complexes that allow the covalent closure of one, two, or all three cohesin interfaces were treated with DMSO or bBBr, denatured with 1% SDS, and separated in 0.45% agarose gel (A), 4%–8% Tris-acetate gels (B), or 0.45% agarose gel containing 0.2% SDS (C). Southern blots were hybridized with α³²P-labeled probe (A and C); Smc3-TEVx3-Scc1-HA6 protein was detected on western blots with α-HA antibody (B). (D) Dimers of 26 kb circular or 42 kb linear minichromosomes held together by modified cohesin complexes were treated with DMSO, bBBr, or BMOE, denatured in 1% SDS, separated in 0.45% agarose gel containing 0.2% SDS, and detected by Southern blotting.

Figure 5

Maintenance but Not Establishment of SCI Depends on Cohesion (A) Wild-type or mutant strains were arrested with α factor pheromone and released into rich media containing nocodazole for 1.5 hr at 37°C. Separation of monomeric and dimeric large circular minichromosomes extracted from wild-type cells (K16150) is shown (A). SCC1's inactivation due to growth of scc1-73 strain (K17889) at restrictive temperature causes complete depletion of dimeric minichromosome species (B). ECO1 inactivation by growth of eco1-1 strain (K17888) at restrictive temperature depletes dimeric minichromosomes (C). Concomitant inactivation of SCC1 and TOP2 proteins by growing the scc1-73 top2-4 double mutant strain (K17892) at restrictive temperature mimics the inactivation of TOP2 protein alone, ensuing total lack of monomeric DNAs (D).

Figure 6

SCI Is Largely Unaffected by Condensin and the Smc5/6 Complex (A) Wild-type or mutant strains were arrested with α factor pheromone and released into rich media containing nocodazole for 1.5 hr at 37°C. Separation of monomeric and dimeric large circular minichromosomes extracted from wild-type cells (K16150) is shown in (A). (B) SCC2's inactivation due to growth of scc2-4 strain (K16149) at restrictive temperature causes complete depletion of dimeric minichromosome species. (C and D) Separation of monomeric and dimeric large circular minichromosomes extracted from smc2-8 (K17893) or smc6-9 (K17895) strains grown at restrictive temperature to induce the inactivation of the SMC2 (C) or SMC6 (D) proteins, respectively.

Figure 7

Cohesin Holds Sister DNAs Together Independent of SCIs (A and B) Yeast strains (K18072) having Cdc20 under the control of the repressible Met3 promoter (Met3P-Cdc20) and bearing the 26 kb circular minichromosome were arrested with α factor pheromone in −Met-TrpRaff medium and released into YPD + 200 mM methionine with or without nocodazole. Minichromosome cohesion was assessed in the presence (B) or absence (A) of microtubule forces on sister kinetochores. (C) DNA catenation throughout the cell cycle. During DNA replication (Rep), SCIs arise concurrently with cohesion establishment. Topo II-driven decatenation takes place throughout S and G2 phases, but complete decatenation is hampered by cohesin's embrace of sister DNAs. Following separase-mediated cohesin removal, Topo II resolves the residual DNA catenanes ensuing complete sister separation.