title: A CENP-S/X complex assembles at the centromere in S and G2 phases of the human cell cycle.
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.
authors: Molecular Biology, FLI, Beutenbergstrasse 11, Jena 07745, Germany.
authors: Carsten
authors: C
authors: Dornblut
authors: Nadine
authors: N
authors: Quinn
authors: Shamci
authors: S
authors: Monajambashi
authors: Lisa
authors: L
authors: Prendergast
authors: Chelly
authors: C
authors: van Vuuren
authors: Sandra
authors: S
authors: Münch
authors: Wen
authors: W
authors: Deng
authors: Heinrich
authors: H
authors: Leonhardt
authors: M Cristina
authors: MC
authors: Cardoso
authors: Christian
authors: C
authors: Hoischen
authors: Stephan
authors: S
authors: Diekmann
authors: Kevin F
authors: KF
authors: Sullivan
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D045744:Cell Line, Tumor; D002503:Centromere / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D016195:G2 Phase; D006367:HeLa Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D016196:S Phase; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; C493243:CENPS protein, human; C551977:CENPX protein, human; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; D004247:DNA
keywords: centromere; constitutive centromere-associated network; kinetochore; mitosis