In vitro loading of human cohesin on DNA by the human Scc2-Scc4 loader complex Journal Article
| Authors: | Bermudez, V. P.; Farina, A.; Higashi, T. L.; Du, F.; Tappin, I.; Takahashi, T. S.; Hurwitz, J. |
| Article Title: | 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 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. |
| Keywords: | unclassified drug; nonhuman; cohesin; animal cell; animals; cell cycle proteins; chromosomal proteins, non-histone; cell cycle; complex formation; sister chromatid; protein stability; in vivo study; in vitro study; dna; double stranded dna; protein purification; chromatin; antibody specificity; dimerization; protein subunit; xenopus; extraction; cell-cycle; dna protein complex; sister chromatid cohesion; cohesin stability; cohesion establishment; cohesin 2; cohesin 4 |
| Journal Title: | Proceedings of the National Academy of Sciences of the United States of America |
| Volume: | 109 |
| Issue: | 24 |
| ISSN: | 0027-8424 |
| Publisher: | National Academy of Sciences |
| Date Published: | 2012-01-01 |
| Start Page: | 9366 |
| End Page: | 9371 |
| Language: | English |
| DOI: | 10.1073/pnas.1206840109 |
| PROVIDER: | scopus |
| PUBMED: | 22628566 |
| PMCID: | PMC3386075 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 2 July 2012" - "CODEN: PNASA" - "Source: Scopus" |
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