A positively charged channel within the Smc1/Smc3 hinge required for sister chromatid cohesion

Abstract

Cohesin's structural maintenance of chromosome 1 (Smc1) and Smc3 are rod-shaped proteins with 50-nm long intra-molecular coiled-coil arms with a heterodimerization domain at one end and an ABC-like nucleotide-binding domain (NBD) at the other. Heterodimerization creates V-shaped molecules with a hinge at their centre. Inter-connection of NBDs by Scc1 creates a tripartite ring within which, it is proposed, sister DNAs are entrapped. To investigate whether cohesin's hinge functions as a possible DNA entry gate, we solved the crystal structure of the hinge from Mus musculus, which like its bacterial counterpart is characterized by a pseudo symmetric heterodimeric torus containing a small channel that is positively charged. Mutations in yeast Smc1 and Smc3 that together neutralize the channel's charge have little effect on dimerization or association with chromosomes, but are nevertheless lethal. Our finding that neutralization reduces acetylation of Smc3, which normally occurs during replication and is essential for cohesion, suggests that the positively charged channel is involved in a major conformational change during S phase.

Figure 1

The M. musculus heterodimeric Smc1/Smc3 hinge domain is structurally similar to the bacterial T. maritima SMC hinge homodimer. (A) Stereo overlay in ribbon depiction of the M. musculus Smc1 (red) and Smc3 (blue) hinge domain structure with the bacterial T. maritima SMC hinge domain (grey). (B) Cartoon representation of the M. musculus Smc1/Smc3 hinge domain. (C) Surface depictions of the M. musculus Smc1/Smc3 hinge domains with electrostatic potentials mapped onto the surface, showing the central channel through the molecule and the highly charged nature of the channel. Images shown are 90° rotations about the x and y axis.

Figure 2

The positively charged central channel is highly conserved in both prokaryotes and eukaryotes. (A) Top row of structures shows the electrostatic potentials mapped onto the surfaces of the SMC homodimer hinge domains of T. maritima (from X-ray structure), B. subtilis and a Halobacterium species (both from in silico models), revealing a conserved and highly positively charged channel. The bottom row of structures reveals the same highly conserved positive charges within the channels of the M. musculus Smc1/Smc3 hinge domain (from X-ray structure), and Drosophila melanogaster and Strongylocentrotus purpuratus (from in silico models). (B) The wild-type residues affected by the five mutations (K554D, K661D in Smc1 (Smc1DD); R665A, K668A, R669A in Smc3 (Smc3AAA)) shown in yellow are mapped onto a cartoon depiction of the model of the S. cerevisiae Smc1/Smc3 hinge domain complex. The structures showing electrostatic potentials on the right reveal the highly positively charged channel in the wild-type protein (top), and the large reduction in charge within the channel for the mutant protein (bottom).

Figure 3

Charge neutralization of the hinge channel does not affect hinge dimerization. (A) Smc1DD and Smc3AAA hinge proteins form stable dimers. Smc1 and Smc3 hinges were either injected as monomers or in an equimolar ratio, separated by size-exclusion chromatography, and fractions analysed by SDS–PAGE. After co-incubation for 10 min at 25°C before injection, Smc1 and Smc3 wild-type proteins (left panel) or Smc1DD and Smc3AAA proteins (right panel) form dimers, resulting in earlier elution of the protein fraction compared with monomeric Smc1 and Smc3 proteins. (B) Smc1DD and Smc3AAA interact tightly. Smc1/Smc3 association constants were determined by ITC. Changes of heat on successive injections of 10 μl Smc3 (100 μM) in a sample cell containing Smc1 (10 μM) were recorded. The peaks were integrated, normalized to the Smc3 concentration and plotted against the molar ratio of Smc3 to Smc1 protein. Data were fitted using a nonlinear least squares fit to a single-site binding model. The K_d for Smc1 and Smc3 wild-type protein binding is 22 nM (left panel). The K_d for Smc1DD and Smc3AAA hinge domain association is 29 nM (middle panel) and 24 nM for Smc1 wild-type and Smc3AAA (right panel). (C) Smc1DD-myc9 competes efficiently with endogenous Smc1 for Smc3AAA-HA3 binding in yeast cell extracts. Control strain (K15426; SMC1-myc9, SMC3-HA3, SMC1, Δsmc3; left lane), channel mutant strain (K15423; smc1DD-myc9, smc3AAA-HA3, SMC1, Δsmc3; middle lane) and an untagged strain (K11850; right lane) were grown exponentially, cells were lysed and protein immunoprecipitated with anti-HA-beads. Beads were washed, boiled and proteins were analysed by SDS–PAGE and visualized by silver staining.

Figure 4

Smc1DD and Smc3AAA form a more stable hinge heterodimer than wild-type Smc1 and Smc3 protein. (A) Smc1-SNAP binds anti-FLAG beads unspecifically. A solution of Smc1 (50 nM), Smc1-SNAP (750 nM) or Smc1DD (50 nM) proteins was added to BSA-blocked anti-FLAG beads. Beads were incubated for 10 min at 16°C and washed quickly three times. Samples were boiled and run on a 10% SDS–PAGE, transferred by western blotting, and anti-HIS antibody was used to detect Smc proteins. Asterisks indicate the heavy chain of IgG from bead-coupled anti-FLAG antibodies. (B) Smc1 binds to Smc3 with a half time of ∼30 min. Smc1 and Smc3-FLAG monomeric hinge proteins were mixed in an equimolar ratio (1 μM each) and incubated for 15 min at 16°C. Smc1-SNAP competitor (final concentration 750 nM) was then added to the pre-bound Smc1/Smc3-FLAG mix (final concentration 50 nM) and incubated at 16°C with shaking. An aliquot of this mix was added every 15 min for 90 min to BSA-blocked anti-FLAG beads. Beads were then incubated for 10 min and washed quickly three times. Samples were boiled and run on a 10% SDS–PAGE, transferred by western blotting, and anti-HIS antibody was used to detect Smc proteins. (C–F) Experiments were performed as described in B, but with Smc1/Smc3AAA-FLAG proteins (C), Smc1DD/Smc3-FLAG proteins (D), Smc1DD/Smc3AAA-FLAG proteins (E) or Smc1M665R/Smc3-FLAG proteins (F). IN, Input; FT, Flow through; B, Bound fraction.

Figure 5

Channel-neutralizing mutations do not affect cohesin's chromosomal distribution genome wide. (A) Genome-wide distribution of Smc1-myc9 and Smc1DD-myc9. Cell extracts of cycling cells (K11850; SMC1-myc9, SMC1, SMC3 and K17075; smc1DD-myc9, SMC1, smc3AAA) were used and Smc1-myc9 (Smc1/Smc3) and Smc1DD-myc9 (Smc1DD/Smc3AAA) were immunoprecipitated and processed for ChIP-seq. Binding ratios of 500 bp running windows (50 bp step size) are shown with red bars. Fold enrichment compared with the WCE is plotted on the y axis in a linear scale. The x axis represents location (kb) along chromosome II. A representative region of 100 kb of chromosome II is depicted (250–150 kb). Average enrichment ratios of mitochondrial and 2 μm DNA were 0.03 and 0.1, respectively, suggesting that all values greater than these represent genuine associations with chromatin. (B) Hydrolysis-defective Smc1E1158Q-myc9 and Smc1DDE1158Q-myc9 bind preferentially to the CEN region. Strains K11857 (smc1E1158Q-myc9, SMC1, SMC3) and K17037 (smc1DDE1158Q-myc9, SMC1, smc3AAA) were prepared and processed as in A.

Figure 6

Channel-neutralized cohesin forms distinct split barrel structures. (A) Smc3-GFP forms a cylindrical structure between marked spindle poles (Cnm67-tdTomato), as observed previously (Yeh et al, 2008) in wild-type cells (K15927; SMC3-GFP, SMC1, SMC3-HA3; left column). The second column represents mutant cells harbouring smc1DD and smc3AAA-GFP mutations (K15947), showing elongated spindles and two cylindrical structures near poles. The third and fourth columns represent the biologically viable single-mutant combinations smc1DD/SMC3-GFP (K16122) and smc3AAA-GFP/SMC1 (K15990), respectively, forming wild-type barrel structures. White arrowheads indicate barrel structure. (B) Spindle pole distances are increased in charge-neutralized mutants. Average spindle pole distance in medium-budded cells for wild-type (K15927) control strain and mutant (K15947, K16122, K15990) strains was plotted. Cnm67-tdTomato was used as spindle pole marker; n=25, P<0.001, error bars=s.d. (C) Neutralization of the hinge channel induces drastic loss of sister chromatid cohesion in metaphase-arrested cells. Wild-type Smc1/Smc3 (K16901; SMC1, SMC3-HA3, smc3-42, ura3::3xURA3 tetO112; tetR-GFP, MET-CDC20) and hinge-neutralized mutant cells Smc1DD/Smc3AAA (K16906; smc1DD, smc3AAA-HA3, smc3-42, ura3::3xURA3 tetO112; tetR-GFP, MET-CDC20) were arrested in metaphase by Cdc20 depletion in Met-containing media for 1 h at 25°C. Cells were then shifted up to 35°C to inactivate smc3-42. After complete arrest (3 h), cells were fixed and sister chromatid cohesion was monitored by Tet operator/repressor-GFP dots at the URA3 locus by fluorescence microscopy; n=100 cells.

Figure 7

Charge neutralization in the hinge channel does not change cohesin's stable association with chromatin, but reduces acetylation of Smc3AAA protein. (A) Smc3AAA-GFP/Smc1DD complex associates stably with chromatin in G2/M. FLIP experiments were performed with a 488 nm laser bleaching the nuclear GFP signal. Fluorescence of the barrel-shaped structure was measured in medium-budded cells of wild-type strain K15927 (SMC3-GFP, SMC1, SMC3-HA3) every 2 s after each bleach pulse for 90 s, and thereafter at every 60 s without bleach pulses for a total of 460 s. Selected images of a single FLIP experiment using Smc3-GFP are shown (right panel). FLIP of Smc3AAA-GFP (K15947; smc3AAA-GFP, smc1DD, SMC3-HA3) in G2/M cells and wild-type Smc3-GFP in post-anaphase cells (K15927) was performed in the same way. Relative fluorescence intensities of Smc3-GFP or Smc3AAA-GFP were plotted over time. Circles indicate bleaching area (n=10, error bars=s.d.). (B) Acetylation of Smc3AAA protein is reduced in charge-neutralized hinge mutant. Crude extract from exponentially growing strains K16270 (SMC1-myc9, SMC3, SMC3-HA3), K15280 (smcDD1-myc9, SMC3, smc3AAA-HA3), K17419 (SMC1-myc9, SMC3, smc3AAA-HA3), K17420 (smc1DD-myc9, SMC3, SMC3-HA3), K15794 (Δrad61/Δeco1, SMC3, SMC3-HA3) and an untagged strain (K15278) were prepared and Smc3-HA3 proteins were immunoprecipitated. Proteins were visualized by western blot using either 3F10 (anti-HA antibody) or antibodies against Smc3-ac. (C) Smc3AAA acetylation is decreased in S phase. Wild-type strain K17328 (SMC1-myc9, SMC3-HA3, SMC3, MET-CDC20) and mutant strain K17329 (smc1DD-myc9, smc3AAA-HA3, SMC3, MET-CDC20) were first arrested in metaphase by Cdc20 depletion in Met-containing medium for 2 h at 30°C. Cells were then released into α-factor-containing medium, arrested for 1 h and again released into Met-containing medium for second metaphase arrest. Samples were taken every 10 min after G1 release and processed for Smc3-HA3 immunoprecipitation. Smc3-HA3 proteins and Smc3-ac proteins were visualized by western blotting using 3F10 and anti-Smc3-ac antibodies, respectively. FACS profile shows cell progression throughout the cell cycle by monitoring DNA content. (D) Deleting the deacetylase HOS1 increases acetylation of Smc3 proteins. Experiment was carried out as in B using strains K17325 (SMC1-myc9, SMC3-HA3, SMC3, HOS1), K17630 (SMC1-myc9, SMC3-HA3, SMC3, Δhos1), K699 (untagged), K17326 (smc1DD-myc9, smc3AAA-HA3, SMC3, HOS1) and K17699 (smc1DD-myc9, smc3AAA-HA3, SMC3, Δhos1). Acetylation increases, in average, by 1.8±0.2-fold (n=3) in Δhos1 cells (SMC1/SMC3, Δhos1) compared with wild-type cells (SMC1/SMC3, HOS1) and by 2.1±0.2-fold (n=3) in Δhos1 channel-neutralized mutant cells (smc1DD/smc3AAA, Δhos1) compared with channel-neutralized mutant cells harbouring the HOS1 gene (smc1DD/smc3AAA, HOS1).

Figure 8

Models of hinge channel function. (A) Model 1. Establishment of cohesion requires first hinge function to allow sister chromatid entrapment. Eco1 acetylates only cohesed sisters, thereby locking them into a stable DNA–protein complex. (B) Model 2. Only when the Smc3 NBD is acetylated, trapping of both sisters occurs by means of the hinge function. (C) Model 3. Cohesin associates with DNA at the onset of S phase by embracing single-stranded DNA or through a stable physical DNA–protein interaction. Cohesin hinge domains open to allow passage of the replisome. Transient association of the hinge domains with the Smc3 NBD stimulates acetylation, which then allows re-shutting of the hinge domains and formation of stable rings around sister chromatids. Acetylation locks cohesin in a stable structural state, possibly with heads disengaged to prevent opening of the ring by ATP hydrolysis.