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

Dornblut, Carsten; Quinn, Nadine; Monajambashi, Shamci; Prendergast, Lisa; Vuuren, Chelly van; Muench, Sandra; Deng, Wen; Leonhardt, Heinrich; Cardoso, M. Cristina; Hoischen, Christian; Diekmann, Stephan und 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, Bd. 4, Nr. 2, 130229

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 Forster 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 (t(1/2) similar to 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.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Biologie > Department Biologie II
• Themengebiete: 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften; Biologie
• ISSN: 2046-2441
• Sprache: Englisch