Mechanical disengagement of the cohesin ring.

Cohesin forms a proteinaceous ring that is thought to link sister chromatids by entrapping DNA and counteracting the forces generated by the mitotic spindle. Whether individual cohesins encircle both sister DNAs and how cohesin opposes spindle-generated forces remains unknown. Here we perform force measurements on individual yeast cohesin complexes either bound to DNA or holding together two DNAs. By covalently closing the hinge and Smc3Psm3-kleisin interfaces we find that the mechanical stability of the cohesin ring entrapping DNA is determined by the hinge domain. Forces of ~20 pN disengage cohesin at the hinge and release DNA, indicating that ~40 cohesin molecules are sufficient to counteract known spindle forces. Our findings provide a mechanical framework for understanding how cohesin interacts with sister chromatids and opposes the spindle-generated tension during mitosis, with implications for other force-generating chromosomal processes including transcription and DNA replication.

SMC complexes: Lifting the lid on loop extrusion.

Loop extrusion has emerged as a prominent hypothesis for how SMC complexes shape chromosomes - single molecule in vitro observations have yielded fascinating images of this process. When not extruding loops, SMC complexes are known to topologically entrap one or more DNAs. Here, we review how structural insight into the SMC complex cohesin has led to a molecular framework for both activities: a Brownian ratchet motion, associated with topological DNA entry, might repeat itself to elicit loop extrusion. After contrasting alternative loop extrusion models, we explore whether topological loading or loop extrusion is more adept at explaining in vivo SMC complex function. SMC variants that experimentally separate topological loading from loop extrusion will in the future probe their respective contributions to chromosome biology.

A Brownian ratchet model for DNA loop extrusion by the cohesin complex.

The cohesin complex topologically encircles DNA to promote sister chromatid cohesion. Alternatively, cohesin extrudes DNA loops, thought to reflect chromatin domain formation. Here, we propose a structure-based model explaining both activities. ATP and DNA binding promote cohesin conformational changes that guide DNA through a kleisin N-gate into a DNA gripping state. Two HEAT-repeat DNA binding modules, associated with cohesin's heads and hinge, are now juxtaposed. Gripping state disassembly, following ATP hydrolysis, triggers unidirectional hinge module movement, which completes topological DNA entry by directing DNA through the ATPase head gate. If head gate passage fails, hinge module motion creates a Brownian ratchet that, instead, drives loop extrusion. Molecular-mechanical simulations of gripping state formation and resolution cycles recapitulate experimentally observed DNA loop extrusion characteristics. Our model extends to asymmetric and symmetric loop extrusion, as well as z-loop formation. Loop extrusion by biased Brownian motion has important implications for chromosomal cohesin function.

When a cell divides, it has to ensure that each of its daughter cells inherits one copy of its genetic information. It does this by duplicating its chromosomes (the DNA molecules that encode the genome) and distributing one copy of each to its daughter cells. Once a cell duplicates a chromosome, the two identical chromosomes must be held together until the cell is ready to divide in two. A ring-shaped protein complex called cohesin does this by encircling the two chromosomes. Cohesin embraces both chromosome copies, as they emerge from the DNA replicating machinery. The complex is formed of several proteins that bind to a small molecule called ATP, whose arrival and subsequent breakdown release energy. Cohesin also interacts with DNA in a different way: it can create loops of chromatin (the complex formed by DNA and its packaging proteins) that help regulate the activity of genes. Experiments performed on single molecules isolated in the laboratory show that cohesin can form a small loop of DNA that is then enlarged through a process called DNA loop extrusion. However, it is not known whether loop extrusion occurs in the cell. Although both of cohesin's roles have to do with how DNA is organised in the cell, it remains unclear how a single protein complex can engage in two such different activities. To answer this question, Higashi et al. used a structure of cohesin from yeast cells gripping onto DNA to build a model that simulates how the complex interacts with chromosomes and chromatin. This model suggested that when ATP is broken down, the cohesin structure shifts and DNA enters the ring, allowing DNA to be entrapped and chromosomes to be bound together. However, a small change in how DNA is gripped initially could prevent it from entering the ring, creating a ratchet mechanism that forms and enlarges a DNA loop. This molecular model helps explain how cohesin can either encircle DNA or create loops. However, Higashi et al.'s findings also raise the question of whether loop extrusion is possible inside cells, where DNA is densely packed and bound to proteins which could be obstacles to loop extrusion. Further research to engineer cohesin that can only perform one of these roles would help to clarify their individual contributions in the cell.

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring.

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

Topological in vitro loading of the budding yeast cohesin ring onto DNA.

The ring-shaped chromosomal cohesin complex holds sister chromatids together by topological embrace, a prerequisite for accurate chromosome segregation. Cohesin plays additional roles in genome organization, transcriptional regulation and DNA repair. The cohesin ring includes an ABC family ATPase, but the molecular mechanism by which the ATPase contributes to cohesin function is not yet understood. Here we have purified budding yeast cohesin, as well as its Scc2-Scc4 cohesin loader complex, and biochemically reconstituted ATP-dependent topological cohesin loading onto DNA. Our results reproduce previous observations obtained using fission yeast cohesin, thereby establishing conserved aspects of cohesin behavior. Unexpectedly, we find that non-hydrolyzable ATP ground state mimetics ADP·BeF2, ADP·BeF3 - and ADP·AlFx, but not a hydrolysis transition state analog ADP·VO4 3-, support cohesin loading. The energy from nucleotide binding is sufficient to drive the DNA entry reaction into the cohesin ring. ATP hydrolysis, thought to be essential for in vivo cohesin loading, must serve a subsequent reaction step. These results provide molecular insight into cohesin function and open new experimental opportunities that the budding yeast model affords.

Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1.

Post-replicative correction of replication errors by the mismatch repair (MMR) system is critical for suppression of mutations. Although the MMR system may need to handle nucleosomes at the site of chromatin replication, how MMR occurs in the chromatin environment remains unclear. Here, we show that nucleosomes are excluded from a >1-kb region surrounding a mismatched base pair in Xenopus egg extracts. The exclusion was dependent on the Msh2-Msh6 mismatch recognition complex but not the Mlh1-containing MutL homologs and counteracts both the HIRA- and CAF-1 (chromatin assembly factor 1)-mediated chromatin assembly pathways. We further found that the Smarcad1 chromatin remodeling ATPase is recruited to mismatch-carrying DNA in an Msh2-dependent but Mlh1-independent manner to assist nucleosome exclusion and that Smarcad1 facilitates the repair of mismatches when nucleosomes are preassembled on DNA. In budding yeast, deletion of FUN30, the homolog of Smarcad1, showed a synergistic increase of spontaneous mutations in combination with MSH6 or MSH3 deletion but no significant increase with MSH2 deletion. Genetic analyses also suggested that the function of Fun30 in MMR is to counteract CAF-1. Our study uncovers that the eukaryotic MMR system has an ability to exclude local nucleosomes and identifies Smarcad1/Fun30 as an accessory factor for the MMR reaction.

The prereplication complex recruits XEco2 to chromatin to promote cohesin acetylation in Xenopus egg extracts.

BACKGROUND: Sister chromatids are held together by the ring-shaped cohesin complex, which is loaded onto chromosomes before DNA replication. Cohesion between sister chromosomes is established during DNA replication, and it requires acetylation of the Smc3 subunit of cohesin by evolutionally conserved cohesin acetyltransferases (CoATs). However, how CoATs are recruited to chromatin and how cohesin acetylation is regulated remain unclear. RESULTS: We found that cohesin acetylation requires pre-RC-dependent chromatin loading of cohesin, but surprisingly, it is independent of DNA synthesis in Xenopus egg extracts. Immunodepletion experiments revealed that XEco2 is the CoAT responsible for Smc3 acetylation and sister chromatid cohesion. Recruitment of XEco2 onto chromatin was dependent on pre-RC assembly but was independent of cohesin loading and DNA synthesis. Two short N-terminal motifs, PBM-A and PBM-B, which are conserved among vertebrate Esco2/XEco2 homologs, were collectively essential for pre-RC-dependent chromatin association of XEco2, cohesin acetylation, and subsequent sister chromatid cohesion. The conserved PCNA-interacting protein box in XEco2 was largely dispensable for Smc3 acetylation but was partially required for cohesion. Interaction of acetylated cohesin with DNA was stabilized against salt-wash treatments after DNA replication. CONCLUSIONS: Our results demonstrate that pre-RC formation regulates chromatin association of XEco2 in Xenopus egg extracts. We propose that this reaction is critical to acetylate cohesin, whose DNA binding is subsequently stabilized by DNA replication.

In vitro loading of human cohesin on DNA by the human Scc2-Scc4 loader complex.

The loading of cohesin onto chromatin requires the heterodimeric complex sister chromatid cohesion (Scc)2 and Scc4 (Scc2/4), which is highly conserved in all species. Here, we describe the purification of the human (h)-Scc2/4 and show that it interacts with h-cohesin and the heterodimeric Smc1-Smc3 complex but not with the Smc1 or Smc3 subunit alone. We demonstrate that both h-Scc2/4 and h-cohesin are loaded onto dsDNA containing the prereplication complex (pre-RC) generated in vitro by Xenopus high-speed soluble extracts. The addition of geminin, which blocks pre-RC formation, prevents the loading of Scc2/4 and cohesin. Xenopus extracts depleted of endogenous Scc2/4 with specific antibodies, although able to form pre-RCs, did not support cohesin loading unless supplemented with purified h-Scc2/4. The results presented here indicate that the Xenopus or h-Scc2/4 complex supports the loading of Xenopus and/or h-cohesin onto pre-RCs formed by Xenopus high-speed extracts. We show that cohesin loaded onto pre-RCs either by h-Scc2/4 and/or the Xenopus complex was dissociated from chromatin by low salt extraction, similar to cohesin loaded onto chromatin in G(1) by HeLa cells in vivo. Replication of cohesin-loaded DNA, both in vitro and in vivo, markedly increased the stability of cohesin associated with DNA. Collectively, these in vitro findings partly recapitulate the in vivo pathway by which sister chromatids are linked together, leading to cohesion.