title: In vitro reconstitution and biochemical analyses of the Schizosaccharomyces pombe nucleosome.
abstract: Schizosaccharomyces pombe, which has a small genome but shares many physiological functions with higher eukaryotes, is a useful single-cell, model eukaryotic organism. In particular, many features concerning chromatin structure and dynamics, including heterochromatin, centromeres, telomeres, and DNA replication origins, are well conserved between S. pombe and higher eukaryotes. However, the S. pombe nucleosome, the fundamental structural unit of chromatin, has not been reconstituted in vitro. In the present study, we established the method to purify S. pombe histones H2A, H2B, H3, and H4, and successfully reconstituted the S. pombe nucleosome in vitro. Our thermal stability assay and micrococcal nuclease treatment assay revealed that the S. pombe nucleosome is markedly unstable and its DNA ends are quite accessible, as compared to the canonical human nucleosome. These findings are important to understand the mechanisms of epigenetic genomic DNA regulation in fission yeast.
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Koyama
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Nagakura
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Tanaka
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Kujirai
authors.affiliation: Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Chikashige
authors.affiliation: Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492, Japan; Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Tokuko
authors.initials: T
authors.lastname: Haraguchi
authors.affiliation: Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492, Japan; Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hiraoka
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; Research Institute for Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; Institute for Medical-oriented Structural Biology, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan. Electronic address: kurumizaka@waseda.jp.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kurumizaka
mesh_terms: D000595:Amino Acid Sequence; D004271:DNA, Fungal / Q000737:chemistry; D006657:Histones / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D009707:Nucleosomes / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012568:Schizosaccharomyces / Q000737:chemistry* / Q000166:cytology; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000302:isolation & purification; D016415:Sequence Alignment
chemical_list: D004271:DNA, Fungal; D006657:Histones; D009707:Nucleosomes; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins
keywords: Histones; Micrococcal nuclease; Nucleosome; Reconstitution; Schizosaccharomyces pombe; Thermal stability