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A CENP-S/X complex assembles at the centromere in S and G2 phases of the human cell cycle.

Dornblut, Carsten and Quinn, Nadine and Monajambashi, Shamci and Prendergast, Lisa and van Vuuren, Chelly and Münch, Sandra and Deng, Wen and Leonhardt, Heinrich and Cardoso, M. Cristina and Hoischen, Christian and Diekmann, Stephan and Sullivan, Kevin F. (2014):
A CENP-S/X complex assembles at the centromere in S and G2 phases of the human cell cycle.
In: Open biology, p. 130229, 4, ISSN 2046-2441,
[Article]

Abstract

The functional identity of centromeres arises from a set of specific nucleoprotein particle subunits of the centromeric chromatin fibre. These include CENP-A and histone H3 nucleosomes and a novel nucleosome-like complex of CENPs -T, -W, -S and -X. Fluorescence cross-correlation spectroscopy and Förster resonance energy transfer (FRET) revealed that human CENP-S and -X exist principally in complex in soluble form and retain proximity when assembled at centromeres. Conditional labelling experiments show that they both assemble de novo during S phase and G2, increasing approximately three- to fourfold in abundance at centromeres. Fluorescence recovery after photobleaching (FRAP) measurements documented steady-state exchange between soluble and assembled pools, with CENP-X exchanging approximately 10 times faster than CENP-S (t1/2 ∼ 10 min versus 120 min). CENP-S binding to sites of DNA damage was quite distinct, with a FRAP half-time of approximately 160 s. Fluorescent two-hybrid analysis identified CENP-T as a uniquely strong CENP-S binding protein and this association was confirmed by FRET, revealing a centromere-bound complex containing CENP-S, CENP-X and CENP-T in proximity to histone H3 but not CENP-A. We propose that deposition of the CENP-T/W/S/X particle reveals a kinetochore-specific chromatin assembly pathway that functions to switch centromeric chromatin to a mitosis-competent state after DNA replication. Centromeres shuttle between CENP-A-rich, replication-competent and H3-CENP-T/W/S/X-rich mitosis-competent compositions in the cell cycle.

Item Type: Article
Erschienen: 2014
Creators: Dornblut, Carsten and Quinn, Nadine and Monajambashi, Shamci and Prendergast, Lisa and van Vuuren, Chelly and Münch, Sandra and Deng, Wen and Leonhardt, Heinrich and Cardoso, M. Cristina and Hoischen, Christian and Diekmann, Stephan and Sullivan, Kevin F.
Title: A CENP-S/X complex assembles at the centromere in S and G2 phases of the human cell cycle.
Language: English
Abstract:

The functional identity of centromeres arises from a set of specific nucleoprotein particle subunits of the centromeric chromatin fibre. These include CENP-A and histone H3 nucleosomes and a novel nucleosome-like complex of CENPs -T, -W, -S and -X. Fluorescence cross-correlation spectroscopy and Förster resonance energy transfer (FRET) revealed that human CENP-S and -X exist principally in complex in soluble form and retain proximity when assembled at centromeres. Conditional labelling experiments show that they both assemble de novo during S phase and G2, increasing approximately three- to fourfold in abundance at centromeres. Fluorescence recovery after photobleaching (FRAP) measurements documented steady-state exchange between soluble and assembled pools, with CENP-X exchanging approximately 10 times faster than CENP-S (t1/2 ∼ 10 min versus 120 min). CENP-S binding to sites of DNA damage was quite distinct, with a FRAP half-time of approximately 160 s. Fluorescent two-hybrid analysis identified CENP-T as a uniquely strong CENP-S binding protein and this association was confirmed by FRET, revealing a centromere-bound complex containing CENP-S, CENP-X and CENP-T in proximity to histone H3 but not CENP-A. We propose that deposition of the CENP-T/W/S/X particle reveals a kinetochore-specific chromatin assembly pathway that functions to switch centromeric chromatin to a mitosis-competent state after DNA replication. Centromeres shuttle between CENP-A-rich, replication-competent and H3-CENP-T/W/S/X-rich mitosis-competent compositions in the cell cycle.

Journal or Publication Title: Open biology
Volume: 4
Divisions: 10 Department of Biology
10 Department of Biology > Cell Biology and Epigenetics
Date Deposited: 08 Apr 2014 12:46
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