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

研究成果: Contribution to journalArticle査読

26 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)9366-9371
ページ数6
ジャーナルProceedings of the National Academy of Sciences of the United States of America
109
24
DOI
出版ステータス出版済み - 6 12 2012

All Science Journal Classification (ASJC) codes

  • General

フィンガープリント 「In vitro loading of human cohesin on DNA by the human Scc2-Scc4 loader complex」の研究トピックを掘り下げます。これらがまとまってユニークなフィンガープリントを構成します。

引用スタイル