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

Abstract

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 hcohesin 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.cohesin stability | cell-cycle | cohesion establishment | sister chromatid cohesion N ewly replicated chromosomes are held together until their separation and equal distribution to daughter cells during cell division. Their association is mediated by cohesin, a foursubunit complex comprising (i) Rad21/sister chromatid cohesion (Scc)1/multiple chloroplast division (Mcd)1, (ii) structural maintenance of chromosomes (Smc)1, (iii) Smc3, and (iv) Scc3/ (stromal antigen) STAG/SA that stably links the sister chromatids together in S and G 2 (1). Smc1 and Smc3 are elongated coiled-coil proteins, each of which folds onto itself to form a globular ATPase head domain through the juxtaposition of the N and C terminus with a hinge domain at the other end. Smc1 and Smc3 interact through their hinge domains to form a heterodimer. The kleisin subunit, Scc1, associates with the head domains of Smc1 and Smc3 to stabilize their interaction and recruits the Scc3/SA subunit. The Smc1-Smc3-Scc1 complex forms a ring structure with an internal diameter of 40 nm, large enough to tether two chromatids (embrace model) (2). Although other models have been proposed to explain how the cohesin ring leads to the association of sister chromatids, substantial evidence supporting the "embrace model" has accumulated (3).Sister chromatid cohesion is a multistep process that includes cohesin loading onto chromatin and the establishment of cohesion during replication (3). Although cohesin loading required for cohesion occurs before the S phase, the timing of its chromosome association differs among species. In ...

Cell cycle-specific cleavage of Scc2 regulates its cohesin deposition activity

“…Vertebrate Scc2 and Scc4 orthologs interact physically through their amino termini (10,13,17,18), but budding yeast Scc2 appears to lack the corresponding amino-terminal region. Nevertheless, we found that Scc2 cleavage prevents its coimmunoprecipitation with Scc4, indicating that the budding yeast Scc2 amino-terminal domain is required for stable interactions with Scc4.…”

“…The amino termini of human and Xenopus Scc2 homologs are required for interactions with their respective Scc4 orthologs (10,13,17,18). Whether this requirement is true in budding yeast is unclear, however, because S. cerevisiae Scc2 lacks the corresponding amino-terminal region present in multicellular eukaryotic homologs.…”

“…because Scc2 and Scc4 protein levels vary little during the cell cycle, or by a lack of assembled cohesin complexes in G 1 , because Scc2/Scc4 chromatin association occurs independently of cohesins (18,27,28). Scc2/Scc4 removal from chromatin is also regulated and occurs during mitosis in Xenopus and, more specifically, during prophase in humans (12,13).…”

“…Interactions of Scc2 and Scc4 orthologs from Xenopus and humans, and their stable association with chromatin, require the amino termini of both proteins (10,13,17,18). In contrast, the fission yeast Scc2 ortholog alone binds nonchromatinized DNA, but does not exhibit an expected preference for sequences shown to associate with Scc2/Scc4 in vivo (19).…”

Nonreplicative functions of the origin recognition complex

“…Evidence indicating an involvement of the pre-RC in the cohesin recruitment to the chromatin has been obtained for other organisms as well. In vitro experiments in X. laevis showed that an extract containing pre-RC provides interaction between the human cohesin complex and DNA, and this interaction may be blocked by inhibitors of the pre-RC assembly [76]. ChIP-seq analysis in D. melanogaster showed that the majority of ORC binding sites coincides with the sites of cohesin localization, thus supporting evidence for the involvement of pre-RC in cohesin recruitment to DNA [77].…”