Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus

Fantahun Diba, Cheryl S. Watson, Bahiru Gametchu

    Research output: Contribution to journalArticle

    47 Citations (Scopus)

    Abstract

    We have examined the targeting of proliferating cell nuclear antigen (PCNA), an integral component of the mammalian replicative enzyme DNA polymerase δ, with sites of DNA replication by using confocal microscopy and computer image analysis. Labeling (5 min pulse) of DNA replication sites in normal human diploid fibroblast cells (NHF1) with BrdU was followed by immunostaining with PCNA antibodies. A striking degree of colocalization was seen between PCNA and the characteristic patterns of DNA replication sites of early, middle and late S-phase (Nakayasu and Berezney [1989] J. Cell. Biol. 108:1-11). These observations were confirmed by quantitative computer image analysis which revealed that approximately 90% of the PCNA-stained area overlapped with DNA replication sites in early S-phase. Pulse-chase experiments, involving in vivo labeling for replication followed by PCNA staining at later time points, suggested that PCNA disassembles from previously replicated sites and targets to newly active sites of DNA replication. To further study this phenomenon in living cells, stable GFP-PCNA transfectants under the control of a tetracycline-inducible promoter were created in mouse 3T6 cells. Like the endogenous PCNA, GFP-PCNA targeted to sites of replication (approximately 80% colocalization) and demonstrated similar dynamic changes following pulse-chase experiments in fixed cells. Studies of living cells revealed progressive changes in the GFP-PCNA distribution that mimic the replication patterns observed in fixed cells. We conclude that GFP-PCNA targets to DNA replication sites in living cells and is an effective marker for tracking the spatio-temporal dynamics of DNA replication as cells transverse the S-phase.

    Original languageEnglish (US)
    Pages (from-to)56-67
    Number of pages12
    JournalJournal of Cellular Biochemistry
    Volume81
    Issue number1
    DOIs
    StatePublished - 2001

    Fingerprint

    Proliferating Cell Nuclear Antigen
    Cell Nucleus
    DNA Replication
    Cells
    DNA
    S Phase
    Labeling
    Image analysis
    Confocal microscopy
    Bromodeoxyuridine
    DNA-Directed DNA Polymerase
    Fibroblasts
    Tetracycline
    Diploidy
    Confocal Microscopy
    Catalytic Domain
    Experiments
    Staining and Labeling

    Keywords

    • Computer image analysis
    • Confocal microscopy
    • DNA replication sites in living cells
    • Green fluorescent protein
    • Normal diploid human fibroblasts

    ASJC Scopus subject areas

    • Biochemistry
    • Cell Biology

    Cite this

    Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus. / Diba, Fantahun; Watson, Cheryl S.; Gametchu, Bahiru.

    In: Journal of Cellular Biochemistry, Vol. 81, No. 1, 2001, p. 56-67.

    Research output: Contribution to journalArticle

    Diba, Fantahun ; Watson, Cheryl S. ; Gametchu, Bahiru. / Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus. In: Journal of Cellular Biochemistry. 2001 ; Vol. 81, No. 1. pp. 56-67.
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    abstract = "We have examined the targeting of proliferating cell nuclear antigen (PCNA), an integral component of the mammalian replicative enzyme DNA polymerase δ, with sites of DNA replication by using confocal microscopy and computer image analysis. Labeling (5 min pulse) of DNA replication sites in normal human diploid fibroblast cells (NHF1) with BrdU was followed by immunostaining with PCNA antibodies. A striking degree of colocalization was seen between PCNA and the characteristic patterns of DNA replication sites of early, middle and late S-phase (Nakayasu and Berezney [1989] J. Cell. Biol. 108:1-11). These observations were confirmed by quantitative computer image analysis which revealed that approximately 90{\%} of the PCNA-stained area overlapped with DNA replication sites in early S-phase. Pulse-chase experiments, involving in vivo labeling for replication followed by PCNA staining at later time points, suggested that PCNA disassembles from previously replicated sites and targets to newly active sites of DNA replication. To further study this phenomenon in living cells, stable GFP-PCNA transfectants under the control of a tetracycline-inducible promoter were created in mouse 3T6 cells. Like the endogenous PCNA, GFP-PCNA targeted to sites of replication (approximately 80{\%} colocalization) and demonstrated similar dynamic changes following pulse-chase experiments in fixed cells. Studies of living cells revealed progressive changes in the GFP-PCNA distribution that mimic the replication patterns observed in fixed cells. We conclude that GFP-PCNA targets to DNA replication sites in living cells and is an effective marker for tracking the spatio-temporal dynamics of DNA replication as cells transverse the S-phase.",
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