Inducible expression of a dominant negative DNA polymerase-γ depletes mitochondrial DNA and produces a ρ0 phenotype

Mona Jazayeri, Alexander Andreyev, Yvonne Will, Manus Ward, Christen M. Anderson*, William Clevenger

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    57 Scopus citations

    Abstract

    We report the inducible, stable expression of a dominant negative form of mitochondria-specific DNA polymerase-γ to eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA polymerase-γ harboring a D1135A substitution (POLGdn). The cells rapidly lost mtDNA (t1/2 = 2-3 days) when expression of the transgene was induced. Concurrent reduction of mitochondrial encoded mRNA and protein, decreased cellular growth rate, and compromised respiration and mitochondrial membrane potential were observed. mtDNA depletion was reversible, as demonstrated by restoration of mtDNA copy number to normal within 10 days when the expression of POLGdn was suppressed following a 3-day induction period. Long term (20 days) expression of POLGdn completely eliminated mtDNA from the cells, resulting in ρ0 cells that were respiration-deficient, lacked electron transport complex activities, and were auxotrophic for pyruvate and uridine. Fusion of the ρ0 cells with human platelets yielded clonal cybrid cell lines that were populated exclusively with donor-derived mtDNA. Respiratory function, mitochondrial membrane potential, and electron transport activities were restored to normal in the cybrid cells. Inducible expression of a dominant negative DNA polymerase-γ can yield mtDNA-deficient cell lines, which can be used to study the impact of specific mtDNA mutations on cellular physiology, and to investigate mitochondrial genome function and regulation.

    Original languageEnglish (US)
    Pages (from-to)9823-9830
    Number of pages8
    JournalJournal of Biological Chemistry
    Volume278
    Issue number11
    DOIs
    StatePublished - Mar 14 2003

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Fingerprint

    Dive into the research topics of 'Inducible expression of a dominant negative DNA polymerase-γ depletes mitochondrial DNA and produces a ρ<sup>0</sup> phenotype'. Together they form a unique fingerprint.

    Cite this