Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo

M Kanemaki, A Sanchez-Diaz, A Gambus, K Labib

    Research output: Contribution to journalArticle

    205 Citations (Scopus)

    Abstract

    Evolutionarily diverse eukaryotic cells share many conserved proteins of unknown function. Some are essential for cell viability(1,2), emphasising their importance for fundamental processes of cell biology but complicating their analysis. We have developed an approach to the large-scale characterization of such proteins, based on conditional and rapid degradation of the target protein in vivo, so that the immediate consequences of bulk protein depletion can be examined(3). Budding yeast strains have been constructed in which essential proteins of unknown function have been fused to a 'heat-inducible-degron' cassette that targets the protein for proteolysis at 37 degreesC (ref. 4). By screening the collection for defects in cell-cycle progression, here we identify three DNA replication factors that interact with each other and that have uncharacterized homologues in human cells. We have used the degron strains to show that these proteins are required for the establishment and normal progression of DNA replication forks. The degron collection could also be used to identify other, essential, proteins with roles in many other processes of eukaryotic cell biology.

    Original languageEnglish
    Pages (from-to)720-724
    Number of pages5
    JournalNature
    Volume423
    Issue number6941
    DOIs
    Publication statusPublished - 12 Jun 2003

    Keywords

    • CDC7
    • YEAST PROTEOME
    • PURIFICATION
    • S-PHASE
    • BUDDING YEAST
    • SACCHAROMYCES-CEREVISIAE
    • FIRING ORIGINS
    • COMPLEXES

    Cite this

    Kanemaki, M ; Sanchez-Diaz, A ; Gambus, A ; Labib, K. / Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo. In: Nature. 2003 ; Vol. 423, No. 6941. pp. 720-724.
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    abstract = "Evolutionarily diverse eukaryotic cells share many conserved proteins of unknown function. Some are essential for cell viability(1,2), emphasising their importance for fundamental processes of cell biology but complicating their analysis. We have developed an approach to the large-scale characterization of such proteins, based on conditional and rapid degradation of the target protein in vivo, so that the immediate consequences of bulk protein depletion can be examined(3). Budding yeast strains have been constructed in which essential proteins of unknown function have been fused to a 'heat-inducible-degron' cassette that targets the protein for proteolysis at 37 degreesC (ref. 4). By screening the collection for defects in cell-cycle progression, here we identify three DNA replication factors that interact with each other and that have uncharacterized homologues in human cells. We have used the degron strains to show that these proteins are required for the establishment and normal progression of DNA replication forks. The degron collection could also be used to identify other, essential, proteins with roles in many other processes of eukaryotic cell biology.",
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    Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo. / Kanemaki, M; Sanchez-Diaz, A; Gambus, A; Labib, K.

    In: Nature, Vol. 423, No. 6941, 12.06.2003, p. 720-724.

    Research output: Contribution to journalArticle

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