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

Vladimir P. Bermudez, Andrea Farina, Torahiko L. Higashi, Fang Du, Inger Tappin, Tatsuro S. Takahashi, Jerard Hurwitz

Research output: Contribution to journalArticle

23 Citations (Scopus)

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 G1 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.

Original languageEnglish
Pages (from-to)9366-9371
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number24
DOIs
Publication statusPublished - Jun 12 2012

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Xenopus
DNA
Chromatin
Chromatids
Geminin
In Vitro Techniques
cohesins
HeLa Cells
Salts
Antibodies

All Science Journal Classification (ASJC) codes

  • General

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In vitro loading of human cohesin on DNA by the human Scc2-Scc4 loader complex. / Bermudez, Vladimir P.; Farina, Andrea; Higashi, Torahiko L.; Du, Fang; Tappin, Inger; Takahashi, Tatsuro S.; Hurwitz, Jerard.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 109, No. 24, 12.06.2012, p. 9366-9371.

Research output: Contribution to journalArticle

Bermudez, Vladimir P. ; Farina, Andrea ; Higashi, Torahiko L. ; Du, Fang ; Tappin, Inger ; Takahashi, Tatsuro S. ; Hurwitz, Jerard. / In vitro loading of human cohesin on DNA by the human Scc2-Scc4 loader complex. In: Proceedings of the National Academy of Sciences of the United States of America. 2012 ; Vol. 109, No. 24. pp. 9366-9371.
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