A positively charged channel within the Smc1/Smc3 hinge required for sister chromatid cohesion
2010; Springer Nature; Volume: 30; Issue: 2 Linguagem: Inglês
10.1038/emboj.2010.315
ISSN1460-2075
AutoresAlexander Kurze, Katharine A. Michie, Sarah E. Dixon, Ajay Mishra, Takehiko Itoh, Syma Khalid, Lana Strmecki, Katsuhiko Shirahige, Christian H. Haering, Jan Löwe, Kim Nasmyth,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoArticle7 December 2010Open Access A positively charged channel within the Smc1/Smc3 hinge required for sister chromatid cohesion Alexander Kurze Alexander Kurze Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Katharine A Michie Katharine A Michie MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Sarah E Dixon Sarah E Dixon Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Ajay Mishra Ajay Mishra Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Takehiko Itoh Takehiko Itoh Laboratory of In Silico Functional Genomics, Graduate School of Bioscience, Tokyo Institute of Technology, Yokohama, Japan Search for more papers by this author Syma Khalid Syma Khalid School of Chemistry, University of Southampton, Southampton, UK Search for more papers by this author Lana Strmecki Lana Strmecki Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Katsuhiko Shirahige Katsuhiko Shirahige Laboratory of Genome Structure and Function, Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Christian H Haering Christian H Haering European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Jan Löwe Jan Löwe MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Kim Nasmyth Corresponding Author Kim Nasmyth Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Alexander Kurze Alexander Kurze Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Katharine A Michie Katharine A Michie MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Sarah E Dixon Sarah E Dixon Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Ajay Mishra Ajay Mishra Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Takehiko Itoh Takehiko Itoh Laboratory of In Silico Functional Genomics, Graduate School of Bioscience, Tokyo Institute of Technology, Yokohama, Japan Search for more papers by this author Syma Khalid Syma Khalid School of Chemistry, University of Southampton, Southampton, UK Search for more papers by this author Lana Strmecki Lana Strmecki Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Katsuhiko Shirahige Katsuhiko Shirahige Laboratory of Genome Structure and Function, Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Christian H Haering Christian H Haering European Molecular Biology Laboratory, Heidelberg, Germany Search for more papers by this author Jan Löwe Jan Löwe MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Kim Nasmyth Corresponding Author Kim Nasmyth Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Author Information Alexander Kurze1, Katharine A Michie2, Sarah E Dixon1, Ajay Mishra1, Takehiko Itoh3, Syma Khalid4, Lana Strmecki1, Katsuhiko Shirahige5, Christian H Haering6, Jan Löwe2 and Kim Nasmyth 1 1Department of Biochemistry, University of Oxford, Oxford, UK 2MRC Laboratory of Molecular Biology, Cambridge, UK 3Laboratory of In Silico Functional Genomics, Graduate School of Bioscience, Tokyo Institute of Technology, Yokohama, Japan 4School of Chemistry, University of Southampton, Southampton, UK 5Laboratory of Genome Structure and Function, Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan 6European Molecular Biology Laboratory, Heidelberg, Germany *Corresponding author. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Tel.: +44 186 561 3229; Fax: +44 186 561 3341; E-mail: [email protected] The EMBO Journal (2011)30:364-378https://doi.org/10.1038/emboj.2010.315 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info 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. Introduction In eukaryotic cells, sister chromatids are held together from their genesis during DNA replication until their disjunction at the metaphase-to-anaphase transition by a complex called cohesin composed of structural maintenance of chromosomes (SMC) proteins Smc1 and Smc3, a kleisin subunit Scc1 (Rad21), Scc3 (SA1/SA2) and a less tightly associated protein called Pds5 (Nasmyth and Haering, 2005). Cohesin's Smc1 and Smc3 subunits fold back on themselves to form 50-nm long rod-shaped intra-molecular anti-parallel coiled coils, with globular 'hinge' domains at one end and an ABC-like nucleotide-binding domain (NBD) at the other end. Heterotypic interactions between Smc1 and Smc3 hinges create stable V-shaped Smc1/Smc3 heterodimers (Melby et al, 1998; Haering et al, 2002; Hirano and Hirano, 2002), which are converted to closed rings by inter-connection of their NBDs by cohesin's Scc1 α-kleisin subunit, whose N- and C-terminal domains bind to Smc3 and Smc1, respectively (Nasmyth and Haering, 2005). In addition to this kleisin-mediated inter-connection, Smc1 and Smc3 NBDs can directly engage in the presence of ATP. Only when engaged in this manner, can ATP molecules sandwiched between Smc1 and Smc3 NBDs be hydrolysed (Arumugam et al, 2006), a process that is essential for cohesin's association with chromosomes (Arumugam et al, 2003; Weitzer et al, 2003). Cohesin's association with chromosomes requires the action of a separate Scc2/Scc4 complex (Ciosk et al, 2000), whereas its ability to connect sister DNAs during S phase requires acetylation of Smc3's NBD by the acetyltransferase Eco1 (Skibbens et al, 1999; Toth et al, 1999; Ivanov et al, 2002; Ben-Shahar et al, 2008; Unal et al, 2008; Rowland et al, 2009). The dissolution of sister chromatid cohesion, which takes place only when all chromosomes have bi-oriented on the mitotic spindle, is triggered by cleavage of cohesin's Scc1 α-kleisin subunit by the thiol protease separase (Uhlmann et al, 1999, 2000; Waizenegger et al, 2000; Hauf et al, 2001). In yeast, most cohesin rings are destroyed by separase (Uhlmann et al, 1999, 2000) and cohesin's re-association with chromosomes depends on re-synthesis of Scc1 shortly before S phase (Michaelis et al, 1997). It has been suggested that cohesin associates stably with chromatin by entrapping DNAs inside its ring (Haering et al, 2002; Gruber et al, 2003). As predicted by this hypothesis, circular sister minichromosomes remain trapped inside rings whose three interfaces have been chemically cross-linked, even after denaturation (Haering et al, 2008). This also implies that cohesion does not involve hitherto uncharacterized interactions between cohesin rings (Zhang et al, 2008). DNA entrapment by cohesin rings presumably entails transient ring opening, that is, the ring must have an entry gate. The finding that cohesin remains functional even after (co-translational) fusion of Smc3 to Scc1 or Scc1 to Smc1 suggests that the entry gate may be situated at the hinge dimerization interface (Gruber et al, 2006). Consistent with this notion is the observation that rapamycin-induced connection of Smc1 and Smc3 hinge domains containing FKBP12 and Frb dimerization domains, respectively, blocks establishment but not maintenance of sister chromatid cohesion (Gruber et al, 2006). To investigate the notion that the Smc1/Smc3 hinge domain has functions besides merely holding Smc1 and Smc3 together, we solved the mouse Smc1/Smc3 hinge crystal structure, which closely resembles the homodimeric bacterial Thermotoga maritima SMC hinge (Haering et al, 2002). In both cases, shallow U-shaped hinge monomers interact to form a pseudo twofold symmetric torus with a small channel in the middle. Remarkably, the channel is positively charged, a feature that, according to modelling, is conserved in SMC hinges from widely different eukaryotic and prokaryotic organisms. We describe a set of amino-acid substitutions in budding yeast Smc1 and Smc3 that, when combined, largely eliminate the channel's positive charge without greatly changing the equilibrium association constant, but reduce the rate of hinge dissociation (Koff) in vitro. Although the neutralizing mutations permit formation of cohesin rings in vivo whose stable association with the genome resembles wild-type cohesin, they drastically reduce Smc3 acetylation and establishment of cohesion during S phase. These data are inconsistent with the notion that Smc1/Smc3 hinges merely act as dimerization domains. They suggest that hinges participate in a major conformational change during S phase, possibly hinge opening, linked to acetylation of Smc3's NBD. Results The structure of the mouse Smc1/Smc3 hinge heterodimer resembles the bacterial SMC hinge homodimer The Mus musculus Smc1/Smc3 hinge heterodimer complex was obtained by co-overexpression of Smc1 residues 471–685 followed by a C-terminal 6xHis tag and Smc3 residues 484–696 in Escherichia coli. We crystallized the Smc1/Smc3 complex and solved its structure using single-wavelength anomalous diffraction data and molecular replacement with the bacterial T. maritima SMC hinge domain structure (Figure 1, Supplementary Tables 1 and 2). Because Smc1 and Smc3 from M. musculus are more similar to each other in primary amino-acid sequence than they are to the T. maritima SMC hinge domain, multi-wavelength anomalous diffraction experiments using SeMet-substituted derivatives of both Smc1 (2-ordered Met) and Smc3 (6-ordered Met) in complex were required to assign each monomer within the heterodimer. The resulting structure was refined to 2.7 Å (Supplementary Table 1). The crystals contained one heterodimer of the Smc1/Smc3 complex, ordered between residues 499 and 675 for Smc1 (chain A), and residues 492 and 670, and 674 and 685 for Smc3 (chain B). 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. Download figure Download PowerPoint The protein fold of the M. musculus Smc1/Smc3 heterodimer resembles the previously published structure of the hinge domain of the SMC homodimer from T. maritima (PDB 1GXL; Haering et al, 2002; Figure 1A). Both N- and C termini of Smc1 and Smc3 hinge domains are present on the same face of the dimer (Figure 1B) and their orientation is consistent with the formation of intra-molecular coiled coils within the 'arms' of the Smc1/Smc3 heterodimers (Haering et al, 2002). Surface representations of the M. musculus hinge domain suggest that there is a small channel running through the centre of the dimerization interface (Figure 1C), as also found in the T. maritima structure. By taking atomic radii into account, the smallest aperture within the channel was determined to be ∼5 Å in diameter, and neither dsDNA nor protein would be able to pass through the channel in the conformation crystallized (Figure 1C, centre). Eukaryotic and prokaryotic SMC hinge channels are positively charged Calculation of the surface electrostatics of the M. musculus Smc1/Smc3 hinge domains reveals that its central channel is highly positively charged, owing to it being lined with many arginines and lysines (Figures 1C and 2B). In silico protein modelling of a large number of prokaryotic SMC complexes (modelled on the T. maritima SMC complex hinge structure) and eukaryotic Smc1/Smc3 hinge domains (modelled on the M. musculus Smc1/Smc3 hinge structure presented here) suggests that the positive charge is a highly conserved feature of the central channel (Figure 2A and Supplementary Figures 1 and 2). Surprisingly, a recently published crystal structure of a conformationally open condensin hinge (mSmc2/mSmc4) revealed rather few positive charges within its inner surface (Griese et al, 2010). The protein used for crystallization, however, lacks a C-terminal β-strand of Smc4 and, as a result, two lysine residues are missing that would point towards the inner surface of the channel. Modelling revealed that an intact and fully closed condensin hinge would also contain a positively charged hinge channel (data not shown). Interestingly, two groups have recently solved the crystal structure of the E. coli MukB homodimeric hinge, which lacks the channel and as a consequence few positive charges are positioned between the dimerization interface (Ku et al, 2010; Li et al, 2010). 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). Download figure Download PowerPoint The positively charged residues in the hinge channel are essential for cohesin's function To investigate the physiological importance of the channel's positive charge, we introduced into Smc1 and Smc3 from Saccharomyces cerevisiae five amino-acid substitutions that together neutralize the charge (Figure 2B and Supplementary Figure 4) without obviously altering the dimerization interface itself. K554 and K661 were mutated to aspartic acid within the Smc1 half hinge (smc1DD), while K668, R665 and R669 were mutated to alanine within the Smc3 half hinge (smc3AAA). In silico modelling shows that only by combining Smc1DD with Smc3AAA is the channel's positive charge largely eliminated (Supplementary Figure 4). Tetrad analysis of spores from a heterozygous SMC1/smc1Δ diploid strain revealed that a single ectopic copy of smc1DD-myc9 fully rescues smc1Δ cells. Similarly, a single ectopic copy of smc3AAA-HA3 fully rescues smc3Δ cells (Supplementary Figure 5A). In contrast, analysis of tetrads from double heterozygous SMC3/smc3Δ SMC1/smc1Δ diploid strains revealed that (unlike their wild-type counterparts) ectopic copies of smc3AAA-HA3 and smc1DD-myc9 together fail to complement smc3Δ smc1Δ double deletion mutants (Supplementary Figure 5A). To investigate the effect on sister chromatid cohesion, we used a strain in which the URA3 locus is marked by the binding of Tet repressor-GFP to multiple tandem Tet operators (Michaelis et al, 1997). Cells expressing the temperature-sensitive smc3-42 allele together with either wild-type or Smc1DD/Smc3AAA proteins were first arrested in metaphase by depleting Cdc20 (for 1 h at 25°C) and then shifted to 35°C for 3 h to inactivate smc3-42. Sister URA3 loci marked by GFP split in only 10% of cells expressing wild-type Smc1/Smc3, but in 70% of cells expressing Smc1DD/Smc3AAA (Figure 6C). We conclude that Smc1DD/Smc3AAA proteins are unable to generate sister chromatid cohesion. 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 Kd for Smc1 and Smc3 wild-type protein binding is 22 nM (left panel). The Kd 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. Download figure Download PowerPoint To test the effect of charge neutralization on cohesin complex formation in vivo, we created yeast strains that express Smc1-myc9 or Smc1DD-myc9, Scc1-PK9 (9xGKPIPNPLLGLDST) and Pds5-PK6 instead of wild-type Smc1, Scc1 and Pds5 proteins, as well as Smc3-HA3 or Smc3AAA-HA3 (ectopic copy) together with endogenous Smc3. Western blotting revealed that immunoprecipitates of Smc3-HA3 or Smc3AAA-HA3 contain similar amounts of Smc1-myc9 or Smc1DD-myc9, Scc1-PK9 (Supplementary Figure 5B) and Pds5-PK6 proteins (Supplementary Figure 5C), suggesting that charge neutralization has little or no effect on cohesin complex formation. Charge-neutralized hinges form stable dimers To address whether the neutralizing mutations weaken Smc1/Smc3 hinge dimerization, we used both theoretical and empirical approaches. We first compared the conformational stability of wild-type and mutant hinges by molecular dynamic (MD) simulations and found little difference on the timescale evaluated (Supplementary Figure 6). We next used size-exclusion chromatography to compare the dimerization properties of purified mutant and wild-type hinges in vitro. Both monomeric wild-type and mutant Smc hinge proteins elute as single peaks, at approximately 13–14 ml, during analytical size-exclusion chromatography (Figure 3A), which suggests that the neutralizing mutations do not adversely affect folding. In contrast, 1:1 molar ratio mixtures of wild-type Smc1 and Smc3 hinge proteins or Smc1DD and Smc3AAA hinge proteins elute as single peaks shifted to 12 ml (Figure 3A). According to this criterion, the channel-neutralizing mutations have little, if any, adverse effect on dimerization. This was confirmed by isothermal titration calorimetry (ITC), which showed that the dissociation constants (Kd) of wild-type (Smc1/Smc3) and mutant (Smc1DD/Smc3AAA) hinges are 22 and 29 nM, respectively (Figure 3B, left and middle panels). ITC did reveal a 4 kcal/mol reduction in the enthalpy of binding, an effect that cannot per se be responsible for the lethality of double-mutant hinges, because the same effect was seen when measuring the binding of wild-type Smc1 and mutant Smc3AAA hinges (Figure 3B, right panel)—a combination that is biologically functional. The enthalpy reduction most likely arises from missing electrostatic interactions; indeed, MD simulation revealed a possible salt bridge (between Smc1 D628 and Smc3 K668) that cannot be formed with the Smc3 K668A mutation. To address whether the mutations affect dimerization in the context of full-length Smc proteins, we created a yeast strain expressing, in addition to endogenous Smc1, Smc3-HA3 and Smc1-myc9 from their own promoters at ectopic sites. Under these conditions, Smc1 and Smc1-myc9 compete for binding to a limited amount of Smc3-HA3, which was immunoprecipitated from cell extracts and proteins analysed by SDS–PAGE and silver staining (Figure 3C, left lane). As expected, similar amounts of Smc1-myc9 and endogenous Smc1 associate with Smc3-HA3. Crucially, a very similar result was obtained with a strain expressing Smc3AAA-HA3 and Smc1DD-myc9 from ectopic sites: Smc3AAA-HA3 co-precipitates similar amounts of mutant and wild-type Smc1 (Figure 3C, middle lane). These results imply that Smc1DD competes efficiently with wild-type Smc1 when binding to Smc3AAA, and that the lethality caused by channel-neutralizing mutations cannot be caused by impaired Smc1/Smc3 dimerization. Channel neutralization reduces hinge dissociation Given that Koff divided by Kon gives the dissociation constant (Kd), charge neutralization could, in principle, increase (or reduce) both constants without greatly altering the Kd. According to the ring model, an increase in Koff could have grave consequences on the maintenance of sister chromatid cohesion, because it would facilitate escape of DNAs from their topological entrapment. To measure Koff, we performed a ligand competition assay (Figure 4). Purified monomeric Smc1 and Smc3-FLAG hinges were mixed in an equimolar ratio. After incubation for 10 min, 15 × molar excess of Smc1 competitor protein (Smc1-SNAP) was added and at 15 min intervals, aliquots of this mixture were added to anti-FLAG beads. Control experiments revealed that insignificant amounts of Smc1 hinge are immunoprecipitated by anti-FLAG beads when Smc3-FLAG hinge protein is omitted (Figure 4A). However, owing to required rapid washing steps and its 15 × excess, some Smc1-SNAP bound nonspecifically to anti-FLAG beads. As a consequence, our assay accurately measures the amount of Smc1 associated with Smc3 at different time points, but not the amount of Smc1-SNAP. At the time of competitor addition (t=0), Smc1 and Smc3-FLAG are present in roughly equal proportions in the FLAG immunoprecipitate, indicating efficient binding, but the amount of Smc1 that co-precipitates with Smc3-FLAG gradually declines with time (Figure 4B). Our data suggest that the hinge dimer has a half-life between 15 and 30 min in vitro. Similar results were obtained for the biologically functional Smc1/Smc3AAA-FLAG and Smc1DD/Smc3-FLAG heterodimers (Figure 4C and D). Remarkably, this assay revealed that Smc1DD/Smc3AAA-FLAG heterodimers are much more stable than wild-type or single-mutant heterodimers (Figure 4E), with no detectable hinge dissociation even after 90 min. To validate the assay further, we analysed Smc1M665R, which disrupts the 'north' Smc1/Smc3 interface (Mishra et al, 2010). This mutation greatly reduces the amount of binding even before competitor addition, as well as the stability of complexes (Figure 4F). Our results suggest that lethality caused by combining Smc1 and Smc3 charge-neutralizing mutations is not due to any intrinsic defect in holding Smc1 and Smc3 together. On the contrary, they raise the possibility that a reduction in Koff might instead be responsible. 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. Download figure Download PowerPoint Hinge channel neutralization has little effect on cohesin's genomic distribution We used high-throughput sequencing after chromatin immunoprecipitation (ChIP-seq) to investigate the effect on cohesin's association with chromatin. To do this, we generated strains expressing either Smc1-myc9 or Smc1DD-myc9 proteins at physiological levels from ectopic loci together with either Smc3 or Smc3AAA from endogenous loci. Owing to the lethality associated with hinge channel neutralization, both strains also expressed untagged Smc1 from the endogenous locus, ensuring viability of both strains. Myc-tagged proteins were immunoprecipitated from exponentially grown cultures after formaldehyde treatment and DNA fragmentation. After sequencing and mapping reads, visual inspection revealed no major differences between the distributions of wild-type and mutant cohesin complexes (Figure 5A), a conclusion confirmed by a scatter plot of Smc1-myc9 versus Smc1DD-myc9 (Supplementary Figure 7A, left panel, Supplementary Figure 7B, top panel). ChIP–qPCR confirmed that there were indeed no major differences in the absolute amount of wild-type and mutant cohesin complexes associated with a variety of loci (Supplementary Figure 7C). The analysis did reveal that Smc1DD-myc9 is slightly more enriched around CEN regions compared with wild-type Smc1-myc9, a conclusion confirmed by ChIP–qPCR (Supplementary Figure 7C, grey and blue bars). 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. Download figure Download PowerPoint To create sister chromatid cohesion, cohesin must be present during DNA replication (Uhlmann and Nasmyth, 1998). In budding yeast, the entire pool of Scc1 is cleaved at the onset of anaphase and cohesin complexes are only re-generated by a burst of Scc1 synthesis shortly before S phase. If the mutations delayed association, then it might occur too late to build cohesion. To address this, ChIP–qPCR was carried out to measure association of wild-
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