The accessory subunit of mitochondrial DNA polymerase γ determines the DNA content of mitochondrial nucleoids in human cultured cells (original) (raw)
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Nucleic Acids Research, 2009
The accessory subunit of mitochondrial DNA polymerase c, POLGb, functions as a processivity factor in vitro. Here we show POLGb has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGb increased nucleoid numbers, whereas overexpression of POLGb reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGb altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGb preferentially bound to plasmids with a short displacement-loop, in contrast to POLGa. These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGb is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2010
Single-stranded DNA binding protein (SSB) plays important roles in DNA replication, recombination and repair through binding to single-stranded DNA. The mammalian mitochondrial SSB (mtSSB) is a bacterial type SSB. In vitro, mtSSB was shown to stimulate the activity of the mitochondrial replicative DNA helicase and polymerase, but its in vivo function has not been investigated in detail. Here we studied the role of mtSSB in the maintenance of mitochondrial DNA (mtDNA) in cultured human cells. RNA interference of mtSSB expression in HeLa cells resulted in rapid reduction of the protein and a gradual decline of mtDNA copy number. The rate of mtDNA synthesis showed a moderate decrease upon mtSSB knockdown in HeLa cells. These results confirmed the requirement of mtSSB for mtDNA replication. Many molecules of mammalian mtDNA hold a short third strand, so-called 7S DNA, whose regulation is poorly understood. In contrast to the gradual decrease of mtDNA copy number, 7S DNA was severely reduced upon mtSSB knockdown in HeLa cells. Further, 7S DNA synthesis was significantly affected by mtSSB knockdown in an oseteosarcoma cell line. These data together suggest that mtSSB plays an important role in the maintenance of 7S DNA alongside its role in mtDNA replication. In addition, live-cell staining of mtDNA did not imply alteration in the organisation of mitochondrial nucleoid protein-mtDNA complexes upon mtSSB knockdown in HeLa cells. This result suggests that the presence of 7S DNA is not crucial for the organisation of mitochondrial nucleoids.
Nucleic Acids Research, 1996
Mitochondria are essential organelles in all eukaryotic cells where cellular ATP is generated through the process of oxidative phosphorylation. Protein components of the respiratory assembly are gene products of both mitochondrial and nuclear genes. The mitochondrial genome itself encodes several protein and nucleic acid components required for such oxidative phosphorylative processes, but the vast majority of genes encoding respiratory chain components are nuclear. Similarly, the processes of replication and transcription of mitochondrial DNA rely exclusively upon RNA and protein species encoded by nuclear genes. We have analyzed two key nuclear-encoded proteins involved in mitochondrial DNA replication and transcription as a function of the presence or absence of mitochondrial DNA. Mitochondrial DNA polymerase (DNA polymerase γ), the nuclear-encoded enzyme which synthesizes mtDNA, is expressed and translated in cells devoid of mitochondrial DNA itself. In contrast, mitochondrial transcription factor A protein levels are tightly linked to the mtDNA status of the cell. These results demonstrate that the DNA polymerase γ protein is stable in the absence of mitochondrial DNA, and that there appears to be no regulatory mechanism present in these cells to alter levels of this protein in the complete absence of mitochondrial DNA. Alternatively, it is possible that this enzyme plays an additional, as yet undefined, role in the cell, thereby mandating its continued production.
Journal of Biological Chemistry, 2000
The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. The D198A amino acid replacement abolished detectable 3-5 (proofreading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous culture. Further culture resulted in the selection of cells with an inactivated mutator polymerase, and a reduced mutation load in mtDNA. Transient expression of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial DNA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression. These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities.
Composition and Dynamics of Human Mitochondrial Nucleoids
Molecular Biology of the Cell, 2003
The organization of multiple mitochondrial DNA (mtDNA) molecules in discrete protein-DNA complexes called nucleoids is well studied in Saccharomyces cerevisiae. Similar structures have recently been observed in human cells by the colocalization of a Twinkle-GFP fusion protein with mtDNA. However, nucleoids in mammalian cells are poorly characterized and are often thought of as relatively simple structures, despite the yeast paradigm. In this article we have used immunocytochemistry and biochemical isolation procedures to characterize the composition of human mitochondrial nucleoids. The results show that both the mitochondrial transcription factor TFAM and mitochondrial single-stranded DNA-binding protein colocalize with Twinkle in intramitochondrial foci defined as nucleoids by the specific incorporation of bromodeoxyuridine. Furthermore, mtDNA polymerase POLG and various other as yet unidentified proteins copurify with mtDNA nucleoids using a biochemical isolation procedure, as does TFAM. The results demonstrated that mtDNA in mammalian cells is organized in discrete protein-rich structures within the mitochondrial network. In vivo time-lapse imaging of nucleoids show they are dynamic structures able to divide and redistribute in the mitochondrial network and suggest that nucleoids are the mitochondrial units of inheritance. Nucleoids did not colocalize with dynaminrelated protein 1, Drp1, a protein of the mitochondrial fission machinery.
In Vitro-Reconstituted Nucleoids Can Block Mitochondrial DNA Replication and Transcription
Cell Reports, 2014
The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication.
DNA polymerases in the mitochondria A critical review of the evidence
Frontiers in Bioscience, 2017
Since 1970, the DNA polymerase gamma (PolG) has been known to be the DNA polymerase responsible for replication and repair of mitochondrial DNA, and until recently it was generally accepted that this was the only polymerase present in mitochondria. However, recent data has challenged that opinion, as several polymerases are now proposed to have activity in mitochondria. To date, their exact role of these other DNA polymerases is unclear and the amount of evidence supporting their