To ensure that each daughter cell receives an equal complement of genetic information, cognate chromatids are paired through replication-coupled sister chromatid cohesion. Cohesion is then actively maintained and eventually enables attachment of kinetochores to the mitotic spindle microtubules emanating from opposite poles to ensure chromosome bi-orientation, prior to subsequent segregation of sister chromatids into daughter cells (Nasmyth and Haering, 2009; Peters and Nishiyama, 2012).

Cohesion is facilitated by cohesin, a member of the Structural Maintenance of Chromosomes (SMC) family of protein complexes, which is responsible for genome organisation across all domains of life (Palecek and Gruber, 2015; Wells et al., 2017). Cohesin complexes form tripartite rings, comprising Smc1-Smc3 and the kleisin subunit Scc1, that are proposed to topologically entrap sister DNA molecules (Gligoris et al., 2014; Nasmyth and Haering, 2009).

The chromosomal addresses of cohesin loading are determined by the Scc2-Scc4 complex, which is enriched at centromeres via direct contacts with kinetochore proteins and promotes DNA-stimulated ATP hydrolysis by the Smc1-Smc3 ATPase heads to drive chromatin entrapment (Ciosk et al., 2000; Hinshaw et al., 2017; Murayama and Uhlmann, 2014). Conversely, dynamic release of DNA from the ring is achieved either by the proteolytic cleavage of the Scc1 kleisin subunit by separase protease (Uhlmann et al., 2000), or by the opening of an ‘exit gate’ formed at the Scc1 and Smc3 interface. Release of the latter is inhibited by Smc3 acetylation and is controlled by the accessory factors Scc3, Wapl and Pds5 (Beckouët et al., 2016; Rolef Ben-Shahar et al., 2008; Rowland et al., 2009; Unal et al., 2008).

Cohesin can associate dynamically with chromatin through alternating cycles of DNA entrapment and release until ring opening is inhibited by acetylation of the Smc3 ATPase head. At this point, DNA is thought to remain topologically entrapped within the SMC-kleisin ring (Gligoris et al., 2014; Beckouët et al., 2016). A prevailing model of genome organisation posits that chromatin loops entrapped within the lumen of the SMC ring are then processively enlarged by extrusion, which presumably forms the basis for higher-order chromatin structure (Alipour and Marko, 2012).

Whereas the essential role of cohesin in modulating genome architecture, gene expression and DNA damage repair is increasingly well established, comparatively little information is available concerning the molecular determinants of its association with chromatin (Busslinger et al., 2017; Haarhuis et al., 2017; Schwarzer et al., 2017; Wu and Yu, 2012). Considerable advances have been made in dissecting the compositional and biochemical prerequisites for topological entrapment of DNA by the cohesin complex (Davidson et al., 2016; Kanke et al., 2016; Stigler et al., 2016; Murayama and Uhlmann, 2014; 2015). However, how cohesin engages its DNA substrate and how such interactions might be regulated is yet to be fully elucidated. Recent work identified a direct DNA-binding site in the paralogous condensin complex, in which the HEAT repeat subunit Ycg1, in complex with the kleisin subunit Brn1, contacts the DNA double helix backbone and stabilizes its association through DNA entrapment within the Brn1 peptide loop (Kschonsak et al., 2017).

To investigate whether cohesin can similarly establish direct contacts with DNA, we produced a series of structurally well-defined globular cohesin subcomplexes and systematically interrogated their ability to bind to DNA. We found that the essential HEAT-repeat subunit Scc3, when in complex with Scc1, binds double-stranded DNA (dsDNA). We further determined the crystal structure of the yeast Scc3-Scc1 complex bound to DNA, which revealed that DNA binding is mediated by a composite surface comprising positively charged amino acid residues from Scc3 and Scc1. Charge-reversal mutagenesis of this interface demonstrates that DNA substrate engagement by the Scc3-Scc1 complex is essential for cohesin to bind to DNA in vitro or chromosomes in vivo and is therefore indispensable for cell viability. Thus, direct DNA substrate engagement through the newly discovered DNA-binding interface of the Scc3-Scc1 subcomplex is a key element for the cohesin cycle.

To investigate the DNA-binding properties of cohesin, we co-expressed and purified defined globular domains and subcomplexes of the cohesin complex from the budding yeast Saccharomyces cerevisiae (Figure 1A, Figure 1—figure supplement 1, Figure 1—figure supplement 2). These encompassed the Smc3 ATPase head domain bound to an N-terminal fragment of Scc1 (Smc3hd-NScc1), the Smc1 ATPase head domain bound to a C-terminal fragment of Scc1 (Smc1hd-CScc1), as well as an Scc3-Scc1 subcomplex (Scc3T-Scc1K) (Figure 1B). In addition, we produced an Smc1-Smc3 hinge heterodimer, Pds5 bound to a Scc1 fragment (Muir et al., 2016) as a full-length (Pds5fl) or truncated variant (Pds5T), as well as Wapl as full-length (Waplfl) or truncated variants (WaplC; Figure 1—figure supplement 1). Consistent with prior studies (Murayama and Uhlmann, 2014), we found that the Scc3T-Scc1K subcomplex and the Smc1-Smc3 hinge heterodimer bound DNA, as seen by the appearance of slower-migrating species in electrophoretic mobility shift assays (EMSAs), as did the Smc3hd-NScc1 module, which has previously been implicated as a DNA sensor in cohesin (Murayama and Uhlmann, 2014) (Figure 1C, Figure 1—figure supplement 2). As expected for non-sequence specific DNA-binding factors, longer DNA fragments (>21 base pairs (bp)) bound more efficiently than shorter DNA duplexes (15 bp) (Figure 1C, Figure 1—figure supplement 2). The ability of the Scc3T-Scc1K subcomplex to bind DNA depended on the presence of Scc1K. Conversely, the other HEAT-repeat-kleisin subcomplex of cohesin, Pds5-Scc1, the Smc1hd-CScc1 subcomplex and the Wapl subunit did not interact with DNA in this assay. Thus, as in the paralogous condensin complex, the HEAT-repeat protein bound to the C-terminal region of the kleisin subunit directly engages DNA (Kschonsak et al., 2017).

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To identify the molecular basis of this interaction, we crystallized the Scc3T-Scc1K complex from budding yeast bound to 19 bp of dsDNA. Optimized crystals diffracted anisotropically to a minimum Bragg spacing of 3.6 Å in the best, and ~5.7 Å in the worst direction (Table 1). We determined the structure by molecular replacement, using the structures of an Scc3 ortholog from Zygosaccharomyces rouxii and a C-terminal fragment of Saccharomyces cerevisiae Scc3 as search models (Roig et al., 2014). The resulting electron-density map provided a continuous trace of the polypeptide main chain, but with a limited level of detail owing to the anisotropy of the data. Despite these drawbacks, we successfully traced the structure using a selenomethionine derivative and refined a model encompassing amino acid residues 134 to 1064 of Scc3 in complex with residues 309 to 400 of Scc1, bound to a 19 bp DNA molecule (Table 1).

As seen in other structures of Scc3, the protein is hook-shaped in the C-terminal section and contains an N-terminal ‘nose’ formed by a pair of extended antiparallel α-helices (Figure 2A). Similarly to the interaction of human Scc1 with Scc3, the yeast Scc1 in our structure binds along the convex surface of the hook-shaped HEAT-repeat subunit. We detected additional electron density corresponding to dsDNA within the cradle of this hook (Figure 2—figure supplement 1A,B). Whereas the DNA duplexes aligned to form pseudocontinuous double helices throughout the crystal, the DNA duplex was slightly too short for tight end-to-end stacking (Figure 2—figure supplement 1A,B). As a result, the DNA density was only partially resolved, apparently due to rotational and translational disorder of the DNA in the binding cavity.

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To identify amino acid residues potentially involved in DNA binding, we mapped the electrostatic surface potential onto the Scc3-Scc1 structure (Figure 2B). This revealed that DNA is nested within an extended cradle spanning the majority of Scc3-Scc1, lined by a set of positively charged residues that directly contact the DNA phosphate backbone. The DNA is aligned almost parallel to the N-terminal ‘nose’ of Scc3, which interacts through a set of positively charged amino acid residues with the DNA of a neighbouring complex related by crystallographic symmetry (Figure 2—figure supplement 1C). We observed no direct nucleotide base–amino acid interactions, which explains the apparent lack of DNA sequence specificity.

To ascertain the amino-acid register of Scc1, we used an anomalous difference map peak for M373 in the selenomethionine-derivative data (Figure 2—figure supplement 1D). The deduced register places Scc1 residue K363 in close proximity to the DNA (Figure 2A, Figure 2—figure supplement 1D). Direct interactions between Scc1 and the DNA phosphate backbone potentially explain why the Scc3T-Scc1K subcomplex has greater DNA binding affinity than does isolated Scc3T (Figure 1C).

Mapping of sequence conservation onto the structure revealed that amino acid residues in the DNA binding groove are generally well conserved among yeast Scc3 orthologs (Figure 3A). In particular, amino acid residues that are located proximal to the DNA phosphate backbone showed strong conservation. To further evaluate the contributions made by individual segments of the DNA-binding surface, we subdivided participating residues into a series of three patches, based on their physical proximity to the DNA (Figure 3B), and subjected these patches to site-directed mutagenesis.

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We measured the DNA equilibrium dissociation constant by fluorescence polarization, using a 32-bp 6-FAM labelled DNA substrate, which is sufficient to bridge the entire DNA binding surface present in the Scc3-Scc1 crystal structure (Figure 3C). Whereas the wild-type Scc3T-Scc1K complex bound this substrate with an equilibrium dissociation constant of 2.2 μM, charge-inversion mutations in patches 1 and 3, located at the crest of and within the Scc3 cradle, had only modest effects on affinity (equilibrium dissociation constants of 9.5 μM and 7.3 μM, respectively). Patch 2 mutants, residing in the Scc3 'nose', exhibited essentially unaltered DNA binding affinity. In contrast, the simultaneous mutation of all three patches (a heptamutant) reduced the binding affinity of the patch 1 or 3 mutants even further (to 29.3 μM, Figure 3C). The defect in binding DNA was not due to any impact of the mutations on the structural integrity of Scc3-Scc1, as all mutant complexes eluted indistinguishably from wild-type Scc3-Scc1 during size-exclusion chromatography (Figure 3—figure supplement 1A) and efficiently formed a complex with Scc1 in vitro (Figure 3—figure supplement 1B) or in vivo (see below). We conclude that the positively charged Scc3 cradle comprises the major DNA-binding surface of Scc3 and that the positively charged amino acid residues located in patch 1 and 3 constitute a composite DNA binding surface. The distribution of these residues across an extended surface of Scc3 might explain why their significance has thus far eluded cell-biological and genetic characterization. To test whether the direct interactions between Scc1 and the DNA phosphate backbone contribute to DNA binding we introduced the double mutation K363E/R364E into Scc1K. This mutant showed an essentially indistinguishable equilibrium dissociation constant as compared to that of the wild-type complex, thus indicating that this positively charged patch of Scc1 does not contribute to DNA binding (Figure 3C).

We then assayed whether DNA binding by Scc3 is important for cohesin function in vivo. We integrated wild-type or mutant versions of Scc3 into a diploid yeast strain in which one of the two endogenous SCC3 genes had been deleted (Supplementary file 2) and analysed the competence of these Scc3 variants to complement the SCC3 deletion by tetrad dissection (Figure 3D). Whereas all three individual patch mutants could support cell proliferation, cells expressing the heptamutant mutant version as their only source of Scc3 failed to divide, despite expressing the mutant Scc3 protein at wild-type levels (Figure 3—figure supplement 2A).

To test whether the inability of the heptamutant version of Scc3 to support cohesin function was the result of a defect in the association of the mutant cohesin complex with chromosomes, we measured the levels of wild-type and mutant cohesin complexes at five independent binding sites in the budding yeast genome by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). The amounts of chromosomal DNA that co-immunoprecipitated with the Scc3 patch 3 mutant were on average 40% lower than those that co-immunoprecipitated with wild-type Scc3. The Scc3 heptamutant failed to bind chromatin entirely (Figure 3E), although it was incorporated normally into cohesin complexes (Figure 3—figure supplement 2B). We conclude that DNA binding by Scc3-Scc1 is essential for the stable association of cohesin complexes with chromosomes in vivo and hence an important determinant of cohesin function.

Targeting of cohesin to the genome is essential for numerous aspects of chromosome biology, including sister chromatid cohesion, DNA damage repair, and transcriptional regulation (Uhlmann, 2016). In this study, we identified a direct DNA-binding site in the Scc3 subunit of the cohesin complex and determined its interaction with DNA at near-atomic resolution. These findings provide evidence for a site of direct DNA contact in cohesin complexes, which presumably contributes to the initial step of chromosome entrapment and/or DNA translocation (Figure 4C).

Figure 4

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We show here that cohesin interacts directly with DNA through a complex formed by the HEAT-repeat subunit Scc3 and the kleisin subunit Scc1. We have determined the DNA-bound structure of Scc3 in complex with a minimal fragment of Scc1 and demonstrated that DNA binding depends on conserved positively charged residues of a composite Scc3-Scc1 interface. We have used this structure to derive DNA-binding deficient Scc3 variants, which fail to support cohesin recruitment to chromatin and consequently cell division. In addition to providing a scaffold for the assembly of cohesin regulators (Hara et al., 2014; Murayama and Uhlmann, 2014; Roig et al., 2014) and thereby participating in the maintenance of cohesion, the Scc3-Scc1 subcomplex also plays a key role in DNA substrate recognition and hence the efficient association of the cohesin holocomplex with chromatin.

The cohesin ring has been proposed to topologically embrace chromosomal DNA, which requires that DNA is loaded into and also released from the ring complex during cohesin’s reaction cycle (Nasmyth, 2011). As mutations in Scc3 that prevent DNA engagement by the Scc3-Scc1 subcomplex in vitro fail to stably bind to chromatin in vivo, it is likely that direct DNA-cohesin interactions contribute to such DNA entry and exit reactions (Figure 4C).

We propose that the Scc3-Scc1 subcomplex provides a dynamic DNA anchoring point that is required for the efficient loading and/or maintenance of cohesin on chromatin. Such a model for Scc3 is supported by previous data, which indicate that Scc3 contributes to cohesin loading (Hu et al., 2011; Murayama and Uhlmann, 2014; Orgil et al., 2015; Roig et al., 2014). In agreement with this model, Scc1 deletion mutants that lack the sequence responsible for binding Scc3 fail to load onto yeast chromosomes (Hu et al., 2011). Furthermore, Scc3 enhances topological DNA entrapment by cohesin in in vitro loading assays (Murayama and Uhlmann, 2014). Thus, DNA binding by the Scc3-Scc1 subcomplex might be the first step in moving chromosomal DNA into the cohesin ring. The subsequent entrapment reaction is then presumably catalysed by the Scc2-Scc4 complex (Davidson et al., 2016; Murayama and Uhlmann, 2014; Stigler et al., 2016).

Surface patch 3 mutations in Scc3, which partially ablate DNA binding by the Scc3-Scc1 subcomplex and reduce cohesin levels on chromatin, do not exhibit any obvious growth defects and are therefore presumably competent to establish sister chromatid cohesion. Indeed, partial depletion of cohesin does not seem to impact some of its core functions, including sister-chromatid cohesion and chromosome segregation (Elbatsh et al., 2016; Heidinger-Pauli et al., 2010). In contrast, even mutations that only slightly impair DNA binding of the equivalent HEAT-repeat/kleisin module of the condensin complex are sufficient to abolish stable chromatin association and to interfere with cell division (Kschonsak et al., 2017). Such discrepancies might be due to alternate loading and/or maintenance mechanisms of cohesin and condensin. Whilst cohesin is loaded by the Scc2-Scc4 complex, no such independent loading factor has been identified for condensin thus far, which could explain why the latter depends more strongly on the direct DNA binding site formed by its HEAT-repeat and kleisin subunits.

Binding of condensin to DNA is further stabilized by the entrapment of the bound DNA helix within a kleisin peptide loop (Figure 4A-) (Kschonsak et al., 2017). The relevant section of Scc1 that would contribute to such topological DNA entrapment is disordered in our structure(Figure 4B). As Scc1 is clearly required for DNA binding of the Scc3-Scc1 subcomplex, but apparently not through direct DNA interactions (Figure 3C), it is possible that cohesin uses a similar mode of chromatin engagement. These findings thus point towards a conserved molecular mechanism that enables chromatin substrate engagement by condensin and cohesin. This mechanism potentially facilitates topological loading, chromatin looping and tracking along chromatin fibres by these SMC complexes (Ganji et al., 2018).

Diffraction data have been deposited in PDB under the accession code 6H8Q.

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Author details

1. Yan Li

   European Molecular Biology Laboratory, Grenoble, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8190-1633

2. Kyle W Muir

   European Molecular Biology Laboratory, Grenoble, France

   Present address

   MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Conceptualization, Formal analysis, Supervision, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   kmuir@mrc-lmb.cam.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0294-5679

3. Matthew W Bowler

   European Molecular Biology Laboratory, Grenoble, France

   Contribution

   Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0465-3351

4. Jutta Metz

   1. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
   2. Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

   Contribution

   Data curation, Methodology

   Competing interests

   No competing interests declared

5. Christian H Haering

   1. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
   2. Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

   Contribution

   Formal analysis, Supervision, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8301-1722

6. Daniel Panne

   Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   panne@embl.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9158-5507

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