Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18

SE Kearsey, S Montgomery, K Labib, K Lindner

    Research output: Contribution to journalArticle

    90 Citations (Scopus)

    Abstract

    We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe, After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. We have applied this method to the DNA replication factor mcm4/cdc21, and find that chromatin association occurs during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication, Upon overexpressing cdc18, we show that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication.

    Original languageEnglish
    Pages (from-to)1681-1690
    Number of pages10
    JournalEMBO Journal
    Volume19
    Issue number7
    DOIs
    Publication statusPublished - 3 Apr 2000

    Keywords

    • GENETIC-ANALYSIS
    • ORIGIN RECOGNITION COMPLEX
    • mcm proteins
    • S-PHASE
    • SCHIZOSACCHAROMYCES-POMBE
    • cell cycle
    • DNA-POLYMERASE-ALPHA
    • NUCLEAR-LOCALIZATION
    • PROTEIN FAMILY
    • BUDDING YEAST
    • cdc18
    • CELL-CYCLE
    • ORC
    • DNA replication
    • SACCHAROMYCES-CEREVISIAE

    Cite this

    @article{4f1a202fd3b4442c83d6afb5a14ea705,
    title = "Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18",
    abstract = "We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe, After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. We have applied this method to the DNA replication factor mcm4/cdc21, and find that chromatin association occurs during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication, Upon overexpressing cdc18, we show that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication.",
    keywords = "GENETIC-ANALYSIS, ORIGIN RECOGNITION COMPLEX, mcm proteins, S-PHASE, SCHIZOSACCHAROMYCES-POMBE, cell cycle, DNA-POLYMERASE-ALPHA, NUCLEAR-LOCALIZATION, PROTEIN FAMILY, BUDDING YEAST, cdc18, CELL-CYCLE, ORC, DNA replication, SACCHAROMYCES-CEREVISIAE",
    author = "SE Kearsey and S Montgomery and K Labib and K Lindner",
    year = "2000",
    month = "4",
    day = "3",
    doi = "10.1093/emboj/19.7.1681",
    language = "English",
    volume = "19",
    pages = "1681--1690",
    journal = "EMBO Journal",
    issn = "0261-4189",
    publisher = "EMBO Press",
    number = "7",

    }

    Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18. / Kearsey, SE; Montgomery, S; Labib, K; Lindner, K.

    In: EMBO Journal, Vol. 19, No. 7, 03.04.2000, p. 1681-1690.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18

    AU - Kearsey, SE

    AU - Montgomery, S

    AU - Labib, K

    AU - Lindner, K

    PY - 2000/4/3

    Y1 - 2000/4/3

    N2 - We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe, After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. We have applied this method to the DNA replication factor mcm4/cdc21, and find that chromatin association occurs during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication, Upon overexpressing cdc18, we show that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication.

    AB - We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe, After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. We have applied this method to the DNA replication factor mcm4/cdc21, and find that chromatin association occurs during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication, Upon overexpressing cdc18, we show that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication.

    KW - GENETIC-ANALYSIS

    KW - ORIGIN RECOGNITION COMPLEX

    KW - mcm proteins

    KW - S-PHASE

    KW - SCHIZOSACCHAROMYCES-POMBE

    KW - cell cycle

    KW - DNA-POLYMERASE-ALPHA

    KW - NUCLEAR-LOCALIZATION

    KW - PROTEIN FAMILY

    KW - BUDDING YEAST

    KW - cdc18

    KW - CELL-CYCLE

    KW - ORC

    KW - DNA replication

    KW - SACCHAROMYCES-CEREVISIAE

    U2 - 10.1093/emboj/19.7.1681

    DO - 10.1093/emboj/19.7.1681

    M3 - Article

    VL - 19

    SP - 1681

    EP - 1690

    JO - EMBO Journal

    JF - EMBO Journal

    SN - 0261-4189

    IS - 7

    ER -