In vitro assembly of physiological cohesin/DNA complexes

Onn, Itay; Koshland, Douglas

doi:10.1073/pnas.1107504108

Cited by 24 publications

(26 citation statements)

References 49 publications

(76 reference statements)

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“…Cohesin located at centromeres appeared dependent on specific kinetochore proteins (28). Possibly in accordance with this notion, studies with crude S-phase yeast extracts reported cohesin loading onto specific pericentric DNA regions (29). The loaded cohesin was stably associated with this DNA (0.5 M KCl).…”

Section: Discussionsupporting

confidence: 63%

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

Bermudez

Farina

Higashi

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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Section: Discussionsupporting

confidence: 63%

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

Bermudez

Farina

Higashi

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…The structural basis through which cohesins maintain sister chromatid pairing remain controversial, but crystal structure analyses of highly conserved SMC complexes provide new insights into this important topic (Box 1). There is also clear evidence that cohesin subsets exhibit different and distinct chromatin-associated dwell times, supporting the notion that cohesin structures cycle between soluble pools and weakly or tightly bound chromatin complexes (Gause et al, 2010;Gerlich et al, 2006;McNairn and Gerton, 2009;Onn and Koshland, 2011). Ascertaining the extent to which altered cohesin conformations impact cohesion-DNA architecture may provide key insights into cohesin function in a wide variety of processes (Fig.…”

Section: Introductionmentioning

confidence: 81%

Cohesin codes – interpreting chromatin architecture and the many facets of cohesin function

Rudra

Skibbens

2013

Journal of Cell Science

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SummarySister chromatid tethering is maintained by cohesin complexes that minimally contain Smc1, Smc3, Mcd1 and Scc3. During S-phase, chromatin-associated cohesins are modified by the Eco1/Ctf7 family of acetyltransferases. Eco1 proteins function during S phase in the context of replicated sister chromatids to convert chromatin-bound cohesins to a tethering-competent state, but also during G2 and M phases in response to double-stranded breaks to promote error-free DNA repair. Cohesins regulate transcription and are essential for ribosome biogenesis and complete chromosome condensation. Little is known, however, regarding the mechanisms through which cohesin functions are directed. Recent findings reveal that Eco1-mediated acetylation of different lysine residues in Smc3 during S phase promote either cohesion or condensation. Phosphorylation and SUMOylation additionally impact cohesin functions. Here, we posit the existence of a cohesin code, analogous to the histone code introduced over a decade ago, and speculate that there is a symphony of posttranslational modifications that direct cohesins to function across a myriad of cellular processes. We also discuss evidence that outdate the notion that cohesion defects are singularly responsible for cohesion-mutant-cell inviability. We conclude by proposing that cohesion establishment is linked to chromatin formation.

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“…DNA sequences per se do not drive cohesin loading (Onn and Koshland, 2011; Murayama and Uhlmann, 2013). Instead, targeting likely depends on interactions between the loading complex and chromatin-associated factors.…”

Section: Discussionmentioning

confidence: 99%

Structural evidence for Scc4-dependent localization of cohesin loading

Hinshaw

Makrantoni

Kerr

et al. 2015

eLife

118

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The cohesin ring holds newly replicated sister chromatids together until their separation at anaphase. Initiation of sister chromatid cohesion depends on a separate complex, Scc2NIPBL/Scc4Mau2 (Scc2/4), which loads cohesin onto DNA and determines its localization across the genome. Proper cohesin loading is essential for cell division, and partial defects cause chromosome missegregation and aberrant transcriptional regulation, leading to severe developmental defects in multicellular organisms. We present here a crystal structure showing the interaction between Scc2 and Scc4. Scc4 is a TPR array that envelops an extended Scc2 peptide. Using budding yeast, we demonstrate that a conserved patch on the surface of Scc4 is required to recruit Scc2/4 to centromeres and to build pericentromeric cohesion. These findings reveal the role of Scc4 in determining the localization of cohesin loading and establish a molecular basis for Scc2/4 recruitment to centromeres.DOI: http://dx.doi.org/10.7554/eLife.06057.001

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In vitro assembly of physiological cohesin/DNA complexes

Cited by 24 publications

References 49 publications

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

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

Cohesin codes – interpreting chromatin architecture and the many facets of cohesin function

Structural evidence for Scc4-dependent localization of cohesin loading

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