title: Stepwise unfolding supports a subunit model for vertebrate kinetochores.
abstract: During cell division, interactions between microtubules and chromosomes are mediated by the kinetochore, a proteinaceous structure located at the primary constriction of chromosomes. In addition to the centromere histone centromere protein A (CENP-A), 15 other members of the constitutive centromere associated network (CCAN) participate in the formation of a chromatin-associated scaffold that supports kinetochore structure. We performed a targeted screen analyzing unfolded centrochromatin from CENP-depleted chromosomes. Our results revealed that CENP-C and CENP-S are critical for the stable folding of mitotic kinetochore chromatin. Multipeak fitting algorithms revealed the presence of an organized pattern of centrochromatin packing consistent with arrangement of CENP-A-containing nucleosomes into up to five chromatin "subunits"-each containing roughly 20-30 nucleosomes. These subunits could be either layers of a boustrophedon or small loops of centromeric chromatin.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, United Kingdom.
authors.forename: Giulia
authors.initials: G
authors.lastname: Vargiu
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, United Kingdom.
authors.forename: Alexandr A
authors.initials: AA
authors.lastname: Makarov
authors.affiliation: MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XU, Scotland, United Kingdom.
authors.forename: James
authors.initials: J
authors.lastname: Allan
authors.affiliation: Laboratory of Chromosome Biology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Fukagawa
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, United Kingdom; Bill.Earnshaw@ed.ac.uk daniel.booth@ed.ac.uk.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Booth
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, United Kingdom; Bill.Earnshaw@ed.ac.uk daniel.booth@ed.ac.uk.
authors.forename: William C
authors.initials: WC
authors.lastname: Earnshaw
mesh_terms: D000818:Animals; D002460:Cell Line; D002503:Centromere / Q000737:chemistry; D000076247:Centromere Protein A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002645:Chickens; D002843:Chromatin / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D018386:Kinetochores / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008938:Mitosis; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry*; D058767:Protein Unfolding; D011993:Recombinant Fusion Proteins; D014714:Vertebrates
chemical_list: D000076247:Centromere Protein A; D002843:Chromatin; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins
keywords: CENP-A; boustrophedon; centrochromatin; centromere; kinetochore