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Biochemical reconstitution of topological DNA binding by the cohesin ring

Nature volume 505, pages 367–371 (2014)Cite this article

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Abstract

Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for faithful chromosome segregation in mitosis. Cohesin also has vital roles in DNA repair and transcriptional regulation. The ring-shaped cohesin complex is thought to encircle sister DNA strands, but its molecular mechanism of action is poorly understood and the biochemical reconstitution of cohesin activity in vitro has remained an unattained goal. Here we reconstitute cohesin loading onto DNA using purified fission yeast cohesin and its loader complex, Mis4Scc2–Ssl3Scc4 (Schizosaccharomyces pombe gene names appear throughout with their more commonly known Saccharomyces cerevisiae counterparts added in superscript). Incubation of cohesin with DNA leads to spontaneous topological loading, but this remains inefficient. The loader contacts cohesin at multiple sites around the ring circumference, including the hitherto enigmatic Psc3Scc3 subunit, and stimulates cohesin’s ATPase, resulting in efficient topological loading. The in vitro reconstitution of cohesin loading onto DNA provides mechanistic insight into the initial steps of the establishment of sister chromatid cohesion and other chromosomal processes mediated by cohesin.

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Figure 1: Mis4Scc2–Ssl3Scc4 is a DNA-binding protein.
Figure 2: In vitro reconstitution of cohesin loading onto DNA.
Figure 3: Topology-mediated DNA binding of cohesin.
Figure 4: Mis4Scc2–Ssl3Scc4 stimulates cohesin’s ATPase.
Figure 5: Mis4Scc2–Ssl3Scc4 interactions around the cohesin ring.

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Acknowledgements

We are grateful to N. O’Reilly for peptide synthesis, A. Alidoust and N. Patel for fermentation and J. Hurwitz, T. Toda and members of the Chromosome Segregation Laboratory for discussion and comments on the manuscript. This work was supported by the European Research Council. Y.M. was supported by the Japanese Society for the Promotion of Science (JSPS).

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Authors and Affiliations

  1. Chromosome Segregation Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK,

    Yasuto Murayama & Frank Uhlmann

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  1. Yasuto Murayama
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  2. Frank Uhlmann
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Contributions

Y.M. designed the study, performed all the experiments, analysed data and wrote the manuscript. F.U. designed and supervised the study and wrote the manuscript.

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Correspondence to Frank Uhlmann.

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Extended data figures and tables

Extended Data Figure 1 Purification and characterization of Mis4Scc2–Ssl3Scc4.

a, Schematic of the Mis4Scc2–Ssl3Scc4 purification. Note that Mis4Scc2 retains a 1 × HA epitope tag at its C terminus after PreScission protease removal of the protein A (ProtA) tag. b, The last purification step of Mis4Scc2–Ssl3Scc4 by size-exclusion chromatography. Proteins were detected by Coomassie brilliant blue (CBB) staining. For comparison, endogenous Mis4Scc2–Ssl3Scc4, containing Mis4Scc2 tagged at its genomic locus, was purified from fission yeast cells. The migration behaviour of the endogenous complex during size-exclusion chromatography was indistinguishable from that of the overexpressed complex. Mis4Scc2–Ssl3Scc4 was detected by silver staining (only Mis4Scc2 is shown). The molecular weight of the complex, based on its hydrodynamic properties during size-exclusion chromatography and gradient centrifugation (compare Fig. 1b), was estimated to be close to the calculated molecular weight of a heterodimer Mis4Scc2–Ssl3Scc4 assembly45. c, The DNA-binding activity co-migrates with Mis4Scc2–Ssl3Scc4 during size-exclusion chromatography. Aliquots of each fraction were analysed by SDS–PAGE followed by CBB staining. Another aliquot was mixed with a 50-nucloetide dsDNA probe and analysed in an electrophoretic mobility shift assay. The gel shift coincided with the Mis4Scc2–Ssl3Scc4 peak during gel filtration. d, The specificity of Mis4Scc2–Ssl3Scc4, and of the Mis4Scc2 subunit alone, for binding to dsDNA was confirmed in a competition assay. In total, 400 nM Mis4Scc2–Ssl3Scc4 or Mis4Scc2 were incubated with 10 nM 32P-labelled 50-nucleotide dsDNA and incubated for 10 min at 37 °C. Non-labelled ssDNA or dsDNA competitor was added to the reaction at the indicated concentrations and incubated for further 10 min before separation by polyacrylamide gel electrophoresis. Gel images are shown, the lower graphs show the quantification. The mean and standard deviation from three independent experiments is shown. DNA binding by Mis4Scc2–Ssl3Scc4 or Mis4Scc2 was efficiently competed by dsDNA but not ssDNA. e, Salt sensitivity of Mis4Scc2–Ssl3Scc4 binding to DNA. 400 nM Mis4Scc2–Ssl3Scc4 (M4S3) or Mis4Scc2 only (M4) was incubated with dsDNA in buffer containing the indicated concentration of NaCl and the electrophoretic mobility shift analysed as above.

Extended Data Figure 2 Purification and characterization of fission yeast cohesin.

a, Diagram of the purification. The last step of the purification, gel filtration of the tetrameric complex consisting of Psm1, Psm3, Rad21 and Psc3, is shown in Fig. 2a. b, ATP-independent DNA binding of fission yeast cohesin. Cohesin was incubated for 15 min at 37 °C in 25 mM Tris/HCl, pH 7.5, 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 50 mM NaCl, 2.5 mM MgCl2, 0.1 mg ml−1 bovine serum albumin (BSA) and 8% (v/v) glycerol, with 0.83 nM of the indicated topologies of the pBluescript KSII (+) plasmid. CCC, covalently closed circular DNA; L, linear DNA; NC, nicked circular DNA. The reactions were separated by 0.8% agarose/Tris-Acetate-EDTA (TAE) gel electrophoresis. DNA was detected by GelRed. This assay revealed topology-independent DNA association of cohesin. c, ATP-independent DNA association of cohesin is salt sensitive. A 5.0-kb dsDNA substrate, corresponding to the ‘N’ sequence (compare Fig. 2g and Methods), was prepared by PCR using biotinylated primers. The PCR product was immobilized on streptavidin-conjugated magnetic beads (Invitrogen). Cohesin was incubated with the DNA beads in 25 mM Tris/HCl, pH 7.5, 1 mM TCEP, 2.5 mM MgCl2, 5% (v/v) glycerol and 0.1% Tween 20, plus the indicated concentrations of KCl. After removing the supernatant fractions, bead-bound proteins were eluted in SDS–PAGE loading buffer and separated by SDS–PAGE followed by CBB staining. Beads without DNA (−DNA) served as a control. d, The purified trimeric cohesin complex consisting of Psm1, Psm3 and Rad21 was analysed by SDS–PAGE next to the wild-type (WT) tetrameric cohesin complex and its variants containing point mutations in the Psm1 or Psm3 Walker A ATPase motif (Psm1WA or Psm3WA) or in both Psm1 and Psm3 loader interaction sites (Psm1/3LM). Purified cohesin containing TEV-cleavable Rad21 (Rad21-TEV) is also shown. Proteins were visualized by CBB staining. e, The Psm1–Psm3 dimer and the Psm3 subunit alone were purified and analysed. f, Purification of his-Psc3, Psc3 and loader interaction site mutant variants, as well as of the Psc3-PkH derivatives, was performed as described in Methods. The proteins were analysed by SDS–PAGE followed by CBB staining.

Extended Data Figure 3 Additional characterization of the in vitro cohesin loading assay.

a, Salt sensitivity of the in vitro cohesin loading reaction. Loading reactions were carried out in the presence of the indicated concentrations of NaCl. A gel image and the quantification of the mean and standard deviation of three independent experiments are shown. b, Requirement of a closed circular DNA substrate for in vitro cohesin loading. Reactions were carried out using the indicated DNA substrates. The mean and standard deviation of three independent experiments are shown. c, The loading reaction facilitated by the Mis4Scc2 subunit alone also results in topological cohesin loading onto DNA. The experiment, including PstI restriction digestion of the assembled cohesin–DNA complexes, was carried out as in Fig. 3b, but using Mis4Scc2 only instead of the Mis4Scc2–Ssl3Scc4 complex. d, Optimization of the Psc3 content in the loading assay. Western blotting with the anti-hexameric histidine antibody suggests that Psc3 is a sub-stoichiometric component in our cohesin preparation (Fig. 2b). This is consistent with the observation that Psc3 is a less stable component of the fission yeast cohesin complex14. Therefore, we analysed whether addition of the individually purified Psc3 subunit improves the in vitro cohesin loading reaction. Shown are reactions in the absence or presence of additional 100 nM his–Psc3, suggesting that Psc3 augments both Mis4Scc2–Ssl3Scc4-dependent and -independent cohesin loading. The graph shows the mean and standard deviation of three independent experiments. Consequently, if not indicated otherwise, we included 100 nM of additional his-Psc3 in all our cohesin loading reactions.

Extended Data Figure 4 Mis4Scc2–Ssl3Scc4 directly interacts with several cohesin subunits.

a, Co-immunoprecipitation of cohesin, Psm1/3 dimer and Psm3 (Extended Data Fig. 2e) with Mis4Scc2–Ssl3Scc4 or Mis4Scc2. 1 out of 20 of the input and the Psm3–Pk immunoprecipitates is shown. Purification of the cohesin subassemblies is shown in Extended Data Fig. 2e. b, Far-western analysis of the Mis4Scc2–Ssl3Scc4 interaction with Psm1 and Psm3. The purified Psm1–Psm3 dimer or Psm3 were separated by SDS–PAGE and transferred to a nitrocellulose membrane. After renaturation, the membrane was probed with Mis4Scc2–Ssl3Scc4, which was then detected using an antibody directed against the HA epitope on Mis4Scc2. BSA served as a control. The data shown in panels a and b suggest that Mis4Scc2–Ssl3Scc4 makes contact with both Psm1 and Psm3. c, Co-immunoprecipitation of Psc3-Pk with Mis4Scc2–Ssl3Scc4 or Mis4Scc2 demonstrates that the cohesin loader directly interacts with the cohesin Psc3 subunit. Purification of Psc3–Pk is shown in Extended Data Fig. 2f.

Extended Data Figure 5 Peptide arrays reveal loader–cohesin interaction sites.

a–c, Peptide array analysis of Mis4Scc2–Ssl3Scc4 interactions with the cohesin subunits Psm3, Rad21 and Psc3 (a, b, and c, respectively). The arrays contain 20-amino-acid-long peptides, tiled with an offset of 2 amino acids, covering the lengths of the three subunits. Numbers on the left indicate the position of the first amino acid in the first peptide in each row. The arrays were probed either with an anti-HA antibody only (control), or with Mis4Scc2–Ssl3Scc4 followed by the anti-HA antibody to detect the HA epitope tag on Mis4Scc2 (+M4S3). Regions of at least three sequential interaction signals (highlighted by boxes) were considered putative interactions sites. Their position on the cohesin complex is summarized in Fig. 5b.

Extended Data Figure 6 Conserved amino acids that mediate cohesin’s interaction with Mis4Scc2–Ssl3Scc4.

a–c, Conservation of Mis4Scc2–Ssl3Scc4 interaction sites in Psm1, Psm3 and Psc3. Two of the Psc3 regions implicated in interaction with Mis4Scc2–Ssl3Scc4 are located within the conserved stromalin domain that is characteristic of Scc3 orthologues. Sequence alignments containing the interaction sites in the three subunits are shown. Bold lines mark the overlap region of interacting peptides on the peptide tiling arrays. Circles highlight amino acid residues that were identified below as important for the interactions and that were further investigated by mutational analysis. The following sequences were included in the alignment: for Psm1, Schizosaccharomyces pombe (Sp) Psm1 (NP_596049.2), Schizosaccharomyces japonicus (Sj) Psm1 (XP_002173070.1), Drosophila melanogaster (Dm) Smc1A (NP_651211.2), Xenopus laevis (Xl) Smc1A (NP_001165905.1), Mus musculus (Mm) Smc1A (NP_062684.1), Homo sapiens (Hs) Smc1A (NP_006297.2); for Psm3, SpPsm3 (NP_593260.1), Sj Psm3 (SJAG05019.4), Dm Smc3 (NP_523374.2), Xl Smc3 (NP_001083742.1), Mm Smc3 (NP_031816.2), Hs Smc3 NP_005436.1); for Psc3, Sp Psc3 (XP_001713063.1), Sp Rec11 (meiosis-specific Psc3 orthologue, NP_588108.3), Saccharomyces cerevisiae Scc3 (NP_ 012238.1), Dm stromalin (NP_477268.2), Xl SA1 (NP_001083864.1), Xl SA2 (NP_001080997.1), Mm SA1 (NP_033308.2), Mm SA2 (NP_067440.3), Mm SA3 (NP_058660.2), Hs SA1 (NP_005853.2), Hs SA2 (NP_006594.3), Hs SA3 (NP_036579.2). d–f, Amino acid scan analysis of Mis4Scc2–Ssl3Scc4-interacting peptides in Psm1, Psm3 and Psc3, respectively. Each position in the 20-amino-acid-long peptides was sequentially substituted with Gly, Leu, Arg, Glu or Tyr. The remaining interaction with Mis4Scc2–Ssl3Scc4 was assessed. Boxes highlight the overlap region of interacting peptides from the peptide tiling array that confines the interaction site. Circles mark amino acid positions that were sensitive to alterations and that were further investigated by mutational analysis.

Extended Data Figure 7 Residues implicated in Mis4Scc2–Ssl3Scc4 interaction are important for cohesin function.

Analysis of phenotypic rescue of temperature sensitive mutations in Psm1 (a), Psm3 (b) and Psc3 (c) by Mis4Scc2–Ssl3Scc4 interaction site mutant subunits. Cohesin subunits bearing the indicated mutations were ectopically expressed under control of their endogenous promoter in temperature-sensitive cohesin mutant fission yeast strains. Complementation of temperature-sensitive growth or sensitivity to the indicated drugs was tested. Western blotting of whole-cell extracts from each strain confirmed that the mutant cohesin subunits were present as stable proteins at comparable levels to the respective wild-type subunits. The Psm1 and Psm3 mutations highlighted in bold were introduced into recombinant cohesin complexes for further analysis. All the Psc3 mutations shown were also included in further biochemical analysis. The strains used were wild type, h− leu1-32 ura4-D18; psm1-897, h− psm1-897 mis4-protein A::kanMX6 leu1-32 ura4-D18; psm3-602, h+ psm3-602; psc3-303, h− psc3-303 cen2+::lacO-ura4+-kanR his7+::Pdis1-GFP-LacI-NLS leu1-32 ura4-D18. In addition to the Psm1, Psm3 and Psc3 subunits, the peptide array analysis also revealed a putative loader interaction with Rad21. Its importance was more difficult to investigate. The region of interaction is less well conserved among species and point mutations in this region led to reduced Rad21 protein stability in fission yeast (data not shown).

Extended Data Figure 8 The role of Psc3 in the cohesin loading reaction.

a, Psc3 is required for Mis4Scc2–Ssl3Scc4-stimulated cohesin loading. Cohesin loading reactions were performed with 150 nM of a cohesin trimer complex lacking Psc3, supplemented with the indicated concentrations of Psc3, or with the cohesin tetramer in the presence or absence of 100 nM Mis4Scc2–Ssl3Scc4. b, Psc3 is required for ATPase stimulation by Mis4Scc2–Ssl3Scc4. The ATPase activity of the cohesin trimer was measured without (−) or with (+) added Psc3 and/or Mis4Scc2–Ssl3Scc4. The results in a and b suggest that Psc3 and the Mis4Scc2–Ssl3Scc4 complex coordinately stimulate ATP-hydrolysis-dependent cohesin loading onto DNA. c, Psc3 variants that are deficient in loader contacts retain functional interactions within the cohesin complex. ATP hydrolysis reactions containing the cohesin trimer complex were supplemented with stoichiometric amounts of wild-type Psc3 or the indicated point mutant variants. The small, but reproducible, stimulation of loader-independent cohesin ATPase activity by Psc3 was unaffected by mutations in loader interaction sites. All panels present mean and standard deviation of three independent experiments.

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Murayama, Y., Uhlmann, F. Biochemical reconstitution of topological DNA binding by the cohesin ring. Nature 505, 367–371 (2014). https://doi.org/10.1038/nature12867

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