Functional Domains of Chicken Mitochondrial Transcription Factor A for the Maintenance of Mitochondrial DNA Copy Number in Lymphoma Cell Line DT40

Matsushima, Yohkazu; Matsumura, Kiyoshi; Ishii, Shinji; Inagaki, Hidetoshi; Suzuki, Tomohiro; Matsuda, Yoichi; Beck, Konrad; Kitagawa, Yasuo

doi:10.1074/jbc.m303842200

Cited by 51 publications

(56 citation statements)

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“…These results indicate that mtDNA is in excess of that needed for normal mitochondrial transcription in Schneider cells. Similar results were obtained upon either suppression or overexpression of the mitochondrial transcription factor A (25,30,31). These data contrast with our finding that mitochondrial transcription factor B2 regulates directly both mtDNA copy number and transcription (25), whereas mitochondrial transcription factor B1 affects neither mtDNA nor transcript levels (26).…”

Section: Discussioncontrasting

confidence: 57%

Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

Matsushima

Kaguni

2007

Journal of Biological Chemistry

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We report the cloning and molecular analysis of Drosophila mitochondrial DNA helicase (d-mtDNA helicase) homologous to human TWINKLE, which encodes one of the genes responsible for autosomal dominant progressive external ophthalmoplegia. An RNA interference construct was designed that reduces expression of d-mtDNA helicase to an undetectable level in Schneider cells. RNA interference knockdown of d-mtDNA helicase decreases the copy number of mitochondrial DNA (mtDNA) ϳ5-fold. In a corollary manner, overexpression of d-mtDNA helicase increases mtDNA levels 1.4-fold. Overexpression of helicase active site mutants K388A and D483A results in a severe depletion of mtDNA and a dominant negative lethal phenotype. Overexpression of mutants analogous to human autosomal dominant progressive external ophthalmoplegia mutations shows differential effects. Overexpression of I334T and A442P mutants yields a dominant negative effect as for the active site mutants. In contrast, overexpression of A326T, R341Q, and W441C mutants results in increased mtDNA copy number, as observed with wild-type overexpression. Our dominant negative analysis of d-mtDNA helicase in cultured cells provides a tractable model for understanding human autosomal dominant progressive external ophthalmoplegia mutations.One of the important functions of mitochondria is the production of ATP by the oxidative phosphorylation pathway. Animal mitochondrial DNA (mtDNA) 2 encodes 13 polypeptides involved in oxidative phosphorylation, whereas all of the factors essential for mtDNA replication are encoded in the nuclear genome (1). In general, mutations in these factors result in both loss of mtDNA integrity via base substitution mutations, duplications, and deletions and mtDNA depletion (2).Autosomal dominant progressive external ophthalmoplegia (adPEO) is a human mitochondrial disorder associated with the presence of multiple deletions of mtDNA (2-5). The disease is manifest in adulthood, typically at 20 -40 years of age; symptoms include muscle weakness, wasting, exercise intolerance, ataxia, hearing loss, cardiomyopathy, and peripheral neuropathy (2). Most of the adPEO families carry heterozygous mutations in one of three genes: ANT1 (the adenine nucleotide translocator 1), POLG (mitochondrial DNA polymerase), and TWINKLE (mtDNA helicase) (2, 6 -8).TWINKLE protein shares high homology with the bacteriophage T7 gene 4 protein (T7 gp4), which contains both helicase and primase catalytic activities located in its carboxyl-and amino-terminal halves, respectively (7). Like other DNA helicases of the DnaB family, T7 gp4 functions as a hexameric ring (9 -11). The amino acid sequence of the helicase domain of T7 gp4 is well conserved in TWINKLE, whereas that of the primase domain is not. TWINKLE exhibits 5Ј-3Ј DNA helicase activity on double-stranded DNA substrates and functions in reconstituted mtDNA replication forks in vitro (12, 13). It co-localizes with mtDNA in structures designated as mitochondrial nucleoids in vivo (7). Recently, TWINKLE was demonstrated to modu...

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Section: Discussioncontrasting

confidence: 57%

Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

Matsushima

Kaguni

2007

Journal of Biological Chemistry

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“…Although the mechanisms by which Lon recognizes its target proteins are not well understood, a recent report showed that E. coli Lon recognizes specific aromatic residue-rich sequences that are hidden in the hydrophobic cores of native structures, but are accessible in unfolded structures (42). Interestingly, the HMG boxes in TFAM contain four conserved aromatic residues within a hydrophobic core, and these residues may be masked when TFAM binds DNA (25,27,29). Another possible explanation is that TFAM not bound to mtDNA becomes exposed to oxidative stress, whereas TFAM bound to mtDNA comprises part of the core of the mitochondrial nucleoids, and is thus surrounded by other proteins (43).…”

Section: Discussionmentioning

confidence: 99%

“…TFAM contains two high mobility group (HMG) amino acid sequence boxes; it binds to mtDNA both specifically and nonspecifically (25). TFAM is essential for mtDNA transcription and for mtDNA packaging in mtDNA maintenance (26)(27)(28)(29)(30). Interestingly, mtDNA and TFAM levels are interdependent, such that knockdown of TFAM results in mtDNA depletion, and reduction of mtDNA copy number causes reduction of TFAM levels (26,29,31).…”

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Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM)

Matsushima

Goto

Kaguni

2010

Proc. Natl. Acad. Sci. U.S.A.

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Lon is the major protease in the mitochondrial matrix in eukaryotes, and is well conserved among species. Although a role for Lon in mitochondrial biogenesis has been proposed, the mechanistic basis is unclear. Here, we demonstrate a role for Lon in mtDNA metabolism. An RNA interference (RNAi) construct was designed that reduces Lon to less than 10% of its normal level in Drosophila Schneider cells. RNAi knockdown of Lon results in increased abundance of mitochondrial transcription factor A (TFAM) and mtDNA copy number. In a corollary manner, overexpression of Lon reduces TFAM levels and mtDNA copy number. Notably, induction of mtDNA depletion in Lon knockdown cells does not result in degradation of TFAM, thereby causing a dramatic increase in the TFAM∶mtDNA ratio. The increased TFAM∶mtDNA ratio in turn causes inhibition of mitochondrial transcription. We conclude that Lon regulates mitochondrial transcription by stabilizing the mitochondrial TFAM∶ mtDNA ratio via selective degradation of TFAM.AAA+ protease | mtDNA maintenance | quality control

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“…Similar to other HMG box proteins, TFAM is able to bind, wrap, bend, and unwind DNA without sequence specificity (10). Furthermore, TFAM is quite abundant and coats mtDNA in Xenopus laevis (11), chicken (12), mouse (13,14), and human cells (15,16). In vivo data from mouse models has demonstrated that disruption of the Tfam gene leads to loss of mtDNA and embryonic lethality (17), whereas increase of TFAM protein levels leads to increase of mtDNA copy number (13).…”

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confidence: 99%

Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA

Kukat

Wurm

Spåhr

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

481

430

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Mammalian mtDNA is packaged in DNA-protein complexes denoted mitochondrial nucleoids. The organization of the nucleoid is a very fundamental question in mitochondrial biology and will determine tissue segregation and transmission of mtDNA. We have used a combination of stimulated emission depletion microscopy, enabling a resolution well below the diffraction barrier, and molecular biology to study nucleoids in a panel of mammalian tissue culture cells. We report that the nucleoids labeled with antibodies against DNA, mitochondrial transcription factor A (TFAM), or incorporated BrdU, have a defined, uniform mean size of approximately 100 nm in mammals. Interestingly, the nucleoid frequently contains only a single copy of mtDNA (average approximately 1.4 mtDNA molecules per nucleoid). Furthermore, we show by molecular modeling and volume calculations that TFAM is a main constituent of the nucleoid, besides mtDNA. These fundamental insights into the organization of mtDNA have broad implications for understanding mitochondrial dysfunction in disease and aging

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Functional Domains of Chicken Mitochondrial Transcription Factor A for the Maintenance of Mitochondrial DNA Copy Number in Lymphoma Cell Line DT40

Cited by 51 publications

References 56 publications

Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM)

Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA

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