title: Dynamics of CENP-N kinetochore binding during the cell cycle.
abstract: Accurate chromosome segregation requires the assembly of kinetochores, multiprotein complexes that assemble on the centromere of each sister chromatid. A key step in this process involves binding of the constitutive centromere-associated network (CCAN) to CENP-A, the histone H3 variant that constitutes centromeric nucleosomes. This network is proposed to operate as a persistent structural scaffold for assembly of the outer kinetochore during mitosis. Here, we show by fluorescence resonance energy transfer (FRET) that the N-terminus of CENP-N lies in close proximity to the N-terminus of CENP-A in vivo, consistent with in vitro data showing direct binding of CENP-N to CENP-A. Furthermore, we demonstrate in living cells that CENP-N is bound to kinetochores during S phase and G2, but is largely absent from kinetochores during mitosis and G1. By measuring the dynamics of kinetochore binding, we reveal that CENP-N undergoes rapid exchange in G1 until the middle of S phase when it becomes stably associated with kinetochores. The majority of CENP-N is loaded during S phase and dissociates again during G2. We propose a model in which CENP-N functions as a fidelity factor during centromeric replication and reveal that the CCAN network is considerably more dynamic than previously appreciated.
authors.affiliation: Molecular Biology, FLI, Beutenbergstrasse 11, 07745 Jena,  Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Hellwig
authors.forename: Stephan
authors.initials: S
authors.lastname: Emmerth
authors.forename: Tobias
authors.initials: T
authors.lastname: Ulbricht
authors.forename: Volker
authors.initials: V
authors.lastname: Döring
authors.forename: Christian
authors.initials: C
authors.lastname: Hoischen
authors.forename: Ronny
authors.initials: R
authors.lastname: Martin
authors.forename: Catarina P
authors.initials: CP
authors.lastname: Samora
authors.forename: Andrew D
authors.initials: AD
authors.lastname: McAinsh
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Carroll
authors.forename: Aaron F
authors.initials: AF
authors.lastname: Straight
authors.forename: Patrick
authors.initials: P
authors.lastname: Meraldi
authors.forename: Stephan
authors.initials: S
authors.lastname: Diekmann
mesh_terms: D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle; D002460:Cell Line; D002503:Centromere / Q000235:genetics / Q000378:metabolism*; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; C571071:CENPN protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone
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