The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities

The TWINKLE protein is a hexameric DNA helicase required for replication of mitochondrial DNA. TWINKLE displays striking sequence similarity to the bacteriophage T7 gene 4 protein (gp4), which is a bi-functional primase-helicase required at the phage DNA replication fork. The N-terminal domain of human TWINKLE contains some of the characteristic sequence motifs found in the N-terminal primase domain of the T7 gp4, but other important motifs are missing. TWINKLE is not an active primase in vitro and the functional role of the N-terminal region has remained elusive. In this report, we demonstrate that the N-terminal part of TWINKLE is required for efficient binding to single-stranded DNA. Truncations of this region reduce DNA helicase activity and mitochondrial DNA replisome processivity. We also find that the gp4 and TWINKLE are functionally distinct. In contrast to the phage protein, TWINKLE binds to double-stranded DNA. Moreover, TWINKLE forms stable hexamers even in the absence of Mg2+ or NTPs, which suggests that an accessory protein, a helicase loader, is needed for loading of TWINKLE onto the circular mtDNA genome

Molecular insights into mitochondrial DNA replication

Mitochondria are organelles found in eukaryotic cells. These organelles produce most of the adenosine triphosphate that cells use as a source of energy. Mitochondria contain their own genomic material, a circular DNA genome (mtDNA) that encodes subunits of the respiratory chain complexes and RNA components needed for mitochondrial translation. Many aspects of mtDNA replication are still not understood and in this thesis we address some of the molecular mechanisms of this process in mammalian cells. DNA synthesis cannot be initiated de novo, but requires a short RNA primer as a starting point. We here demonstrate that the mitochondrial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the origin of light strand DNA replication (OriL) in human mtDNA. Using purified POLRMT and the core factors of the mitochondrial replisome, we faithfully reconstitute OriLdependent initiation of replication in vitro. During origin activation, OriL is exposed in its single-stranded conformation and adopts a stem-loop structure. POLRMT initiates primer synthesis from a poly-dT stretch in the single-stranded loop region and after about 25 nt, POLRMT is replaced by the mitochondrial DNA polymerase ! (POL!) and DNA synthesis is initiated. Our findings also suggest that the mitochondrial single-stranded DNA binding protein directs origin-specific initiation by efficiently blocking unspecific initiation events in other regions of the mtDNA genome. To analyze the requirements of OriL in vivo, we have used saturation mutagenesis in the mouse combined with in vitro biochemistry and demonstrated that OriL is essential for mtDNA maintenance. OriL requires a stable stem-loop structure and a pyrimidine-rich sequence in the template strand for proper origin function. The OriL mechanism appears to be conserved, since bioinformatics analyses demonstrated the presence of OriL in the mtDNA of most vertebrates including birds. Our findings suggest that mtDNA replication may be performed by a common mechanism in all vertebrates and lend support to the strand-displacement model for mtDNA replication. A molecular understanding of the mitochondrial DNA replication machinery is also of medical importance. Today, more than 160 mutations in the gene encoding the catalytic subunit of POL! (POL!A) have been associated with human disease. One example is the Y955C mutation, which causes autosomal dominant progressive external ophthalmoplegia, a disorder characterized by the accumulation of multiple mtDNA deletions. The Y955C mutation decreases POL! processivity due to a decreased binding affinity for the incoming deoxyribonucleoside triphosphate. However, it is not clear why this biochemical defect leads to a dominant disease. We have used the reconstituted mammalian mtDNA replisome and studied functional consequences of the dominant Y955C mutation. Our study revealed that the POL!A:Y955C enzyme is prone to stalling at dATP insertion sites and instead enters a polymerase/exonuclease idling mode. The mutant POL!A:Y955C competes with wild-type POL!A for access to the primer template. However, once assembled in the replisome, the wild-type enzyme is no longer affected. Our data therefore provide a mechanism for the mtDNA replication phenotypes seen in patients harboring the Y955C mutation

In Vitro-Reconstituted Nucleoids Can Block Mitochondrial DNA Replication and Transcription

SummaryThe 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

The mitochondrial DNA helicase TWINKLE can assemble on a closed circular template and support initiation of DNA synthesis

Mitochondrial DNA replication is performed by a simple machinery, containing the TWINKLE DNA helicase, a single-stranded DNA-binding protein, and the mitochondrial DNA polymerase γ. In addition, mitochondrial RNA polymerase is required for primer formation at the origins of DNA replication. TWINKLE adopts a hexameric ring-shaped structure that must load on the closed circular mtDNA genome. In other systems, a specialized helicase loader often facilitates helicase loading. We here demonstrate that TWINKLE can function without a specialized loader. We also show that the mitochondrial replication machinery can assemble on a closed circular DNA template and efficiently elongate a DNA primer in a manner that closely resembles initiation of mtDNA synthesis in vivo

Seuil critique d'hétéroplasmie d'une mutation mitochondriale chez Drosophila subobscura (contrôle nucléaire et mécanismes de compensation biochimiques)

CLERMONT FD-BCIU Sci.et Tech. (630142101) / SudocSudocFranceF

La prestation compensatoire devant les Cours d'appel de Grenoble et Chambéry pour l'année 2016

Restitution de travaux - projet GIP Barémisatio

Organization of DNA in Mammalian Mitochondria

International audienceAs with all organisms that must organize and condense their DNA to fit within the limited volume of a cell or a nucleus, mammalian mitochondrial DNA (mtDNA) is packaged into nucleoprotein structures called nucleoids. In this study, we first introduce the general modes of DNA compaction, especially the role of the nucleoid-associated proteins (NAPs) that structure the bacterial chromosome. We then present the mitochondrial nucleoid and the main factors responsible for packaging of mtDNA: ARS- (autonomously replicating sequence-) binding factor 2 protein (Abf2p) in yeast and mitochondrial transcription factor A (TFAM) in mammals. We summarize the single-molecule manipulation experiments on mtDNA compaction and visualization of mitochondrial nucleoids that have led to our current knowledge on mtDNA compaction. Lastly, we discuss the possible regulatory role of DNA packaging by TFAM in DNA transactions such as mtDNA replication and transcription

Réflexions sur la traduction d’Une gourmandise, de Muriel Barbery

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