Conformational dynamics of cohesin/Scc2 loading complex are regulated by Smc3 acetylation and ATP binding.

The ring-shaped cohesin complex is a key player in sister chromatid cohesion, DNA repair, and gene transcription. The loading of cohesin to chromosomes requires the loader Scc2 and is regulated by ATP. This process is hindered by Smc3 acetylation. However, the molecular mechanism underlying this inhibition remains mysterious. Here, using Saccharomyces cerevisiae as a model system, we identify a novel configuration of Scc2 with pre-engaged cohesin and reveal dynamic conformations of the cohesin/Scc2 complex in the loading reaction. We demonstrate that Smc3 acetylation blocks the association of Scc2 with pre-engaged cohesin by impairing the interaction of Scc2 with Smc3's head. Lastly, we show that ATP binding induces the cohesin/Scc2 complex to clamp DNA by promoting the interaction between Scc2 and Smc3 coiled coil. Our results illuminate a dynamic reconfiguration of the cohesin/Scc2 complex during loading and indicate how Smc3 acetylation and ATP regulate this process.

Folding of cohesin's coiled coil is important for Scc2/4-induced association with chromosomes.

Cohesin's association with and translocation along chromosomal DNAs depend on an ATP hydrolysis cycle driving the association and subsequent release of DNA. This involves DNA being 'clamped' by Scc2 and ATP-dependent engagement of cohesin's Smc1 and Smc3 head domains. Scc2's replacement by Pds5 abrogates cohesin's ATPase and has an important role in halting DNA loop extrusion. The ATPase domains of all SMC proteins are separated from their hinge dimerisation domains by 50-nm-long coiled coils, which have been observed to zip up along their entire length and fold around an elbow, thereby greatly shortening the distance between hinges and ATPase heads. Whether folding exists in vivo or has any physiological importance is not known. We present here a cryo-EM structure of the apo form of cohesin that reveals the structure of folded and zipped-up coils in unprecedented detail and shows that Scc2 can associate with Smc1's ATPase head even when it is fully disengaged from that of Smc3. Using cysteine-specific crosslinking, we show that cohesin's coiled coils are frequently folded in vivo, including when cohesin holds sister chromatids together. Moreover, we describe a mutation (SMC1D588Y) within Smc1's hinge that alters how Scc2 and Pds5 interact with Smc1's hinge and that enables Scc2 to support loading in the absence of its normal partner Scc4. The mutant phenotype of loading without Scc4 is only explicable if loading depends on an association between Scc2/4 and cohesin's hinge, which in turn requires coiled coil folding.

Transport of DNA within cohesin involves clamping on top of engaged heads by Scc2 and entrapment within the ring by Scc3.

In addition to extruding DNA loops, cohesin entraps within its SMC-kleisin ring (S-K) individual DNAs during G1 and sister DNAs during S-phase. All three activities require related hook-shaped proteins called Scc2 and Scc3. Using thiol-specific crosslinking we provide rigorous proof of entrapment activity in vitro. Scc2 alone promotes entrapment of DNAs in the E-S and E-K compartments, between ATP-bound engaged heads and the SMC hinge and associated kleisin, respectively. This does not require ATP hydrolysis nor is it accompanied by entrapment within S-K rings, which is a slower process requiring Scc3. Cryo-EM reveals that DNAs transported into E-S/E-K compartments are 'clamped' in a sub-compartment created by Scc2's association with engaged heads whose coiled coils are folded around their elbow. We suggest that clamping may be a recurrent feature of cohesin complexes active in loop extrusion and that this conformation precedes the S-K entrapment required for sister chromatid cohesion.

Scc2 counteracts a Wapl-independent mechanism that releases cohesin from chromosomes during G1.

Cohesin's association with chromosomes is determined by loading dependent on the Scc2/4 complex and release due to Wapl. We show here that Scc2 also actively maintains cohesin on chromosomes during G1 in S. cerevisiae cells. It does so by blocking a Wapl-independent release reaction that requires opening the cohesin ring at its Smc3/Scc1 interface as well as the D loop of Smc1's ATPase. The Wapl-independent release mechanism is switched off as cells activate Cdk1 and enter G2/M and cannot be turned back on without cohesin's dissociation from chromosomes. The latter phenomenon enabled us to show that in the absence of release mechanisms, cohesin rings that have already captured DNA in a Scc2-dependent manner before replication no longer require Scc2 to capture sister DNAs during S phase.

A Protocol for Assaying the ATPase Activity of Recombinant Cohesin Holocomplexes.

Cohesin and other members of the structural maintenance of chromosomes (SMC)-kleisin family such as condensin and Smc5-6, as well as central players in genome function and structure such as topoisomerases, DNA and RNA polymerases, and DNA repair enzymes contain nucleotide binding domains (NBD) which bind and eventually cleave ATP. The released energy is harnessed in various ways by these enzymes in order to fulfill their essential functions. However, unlike other enzymes, Smc-kleisin complexes-well sized, elongated and multisubunit in nature-have only recently been purified as holocomplexes. This progress offers both the opportunity and the challenge to determine in detail the potency of the ATPase activity of these large protein assemblies-typically exceeding 0.5 MDa in molecular weight-and examine its mechanistic features. We describe here in further detail a combined comprehensive protocol which we have successfully employed before for assaying the ATPase activity of recombinant budding yeast cohesin holocomplexes. We believe that with small and appropriate modifications the methods described here should be applicable to other ATPase complexes.

Scc2 Is a Potent Activator of Cohesin's ATPase that Promotes Loading by Binding Scc1 without Pds5.

Cohesin organizes DNA into chromatids, regulates enhancer-promoter interactions, and confers sister chromatid cohesion. Its association with chromosomes is regulated by hook-shaped HEAT repeat proteins that bind Scc1, namely Scc3, Pds5, and Scc2. Unlike Pds5, Scc2 is not a stable cohesin constituent but, as shown here, transiently replaces Pds5. Scc1 mutations that compromise its interaction with Scc2 adversely affect cohesin's ATPase activity and loading. Moreover, Scc2 mutations that alter how the ATPase responds to DNA abolish loading despite cohesin's initial association with loading sites. Lastly, Scc2 mutations that permit loading in the absence of Scc4 increase Scc2's association with chromosomal cohesin and reduce that of Pds5. We suggest that cohesin switches between two states: one with Pds5 bound that is unable to hydrolyze ATP efficiently but is capable of release from chromosomes and another in which Scc2 replaces Pds5 and stimulates ATP hydrolysis necessary for loading and translocation from loading sites.

The Cohesin Ring Uses Its Hinge to Organize DNA Using Non-topological as well as Topological Mechanisms.

As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion. In most cells where cohesin loads without conferring cohesion, it does so by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen is neutralized loads and moves along chromatin despite failing to entrap DNAs. Thus, cohesin engages chromatin in non-topological, as well as topological, manners. Since hinge mutations, but not Smc-kleisin fusions, abolish entrapment, DNAs may enter cohesin rings through hinge opening. Mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin's recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin's hinge driven by cycles of ATP hydrolysis.