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As 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.

Mammalian mitochondria contain a circular genome (mtDNA) which encodes subunits of the oxidative phosphorylation machinery. The replication and maintenance of mtDNA is carried out by a set of nuclear-encoded factors—of which, helicases form an important group. The TWINKLE helicase is the main helicase in mitochondria and is the only helicase required for mtDNA replication. Mutations in TWINKLE cause a number of human disorders associated with mitochondrial dysfunction, neurodegeneration and premature ageing. In addition, a number of other helicases with a putative role in mitochondria have been identified. In this review, we discuss our current knowledge of TWINKLE structure and function and its role in diseases of mtDNA maintenance. We also briefly discuss other potential mitochondrial helicases and postulate on their role(s) in mitochondria.

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system 1 – 6 . The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS 7 – 17 , and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease. Inhibitors of mitochondrial transcription that target human mitochondrial RNA polymerase provide a chemical biology tool for studying the role of mitochondrial DNA expression in a wide range of pathologies.

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 ∼100 nm in mammals. Interestingly, the nucleoid frequently contains only a single copy of mtDNA (average ∼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.

Mitochondrial DNA (mtDNA) can undergo double-strand breaks (DSBs), caused by defective replication, or by various endogenous or exogenous sources, such as reactive oxygen species, chemotherapeutic agents or ionizing radiations. MtDNA encodes for proteins involved in ATP production, and maintenance of genome integrity following DSBs is thus of crucial importance. However, the mechanisms involved in mtDNA maintenance after DSBs remain unknown. In this study, we investigated the consequences of the production of mtDNA DSBs using a human inducible cell system expressing the restriction enzyme PstI targeted to mitochondria. Using this system, we could not find any support for DSB repair of mtDNA. Instead we observed a loss of the damaged mtDNA molecules and a severe decrease in mtDNA content. We demonstrate that none of the known mitochondrial nucleases are involved in the mtDNA degradation and that the DNA loss is not due to autophagy, mitophagy or apoptosis. Our study suggests that a still uncharacterized pathway for the targeted degradation of damaged mtDNA in a mitophagy/autophagy-independent manner is present in mitochondria, and might provide the main mechanism used by the cells to deal with DSBs.

Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.

Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after ~120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3'-ends that can be used by DNA polymerase γ to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggest a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.

Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.

Non-coding mutations in the TERT promoter ( TERT p), typically at one of two bases −124 and −146 bp upstream of the start codon, are among the most prevalent driver mutations in human cancer. Several additional recurrent TERT p mutations have been reported but their functions and origins remain largely unexplained. Here, we show that atypical TERT p mutations arise secondary to canonical TERT p mutations in a two-step process. Canonical TERT p mutations create de novo binding sites for ETS family transcription factors that induce favourable conditions for DNA damage formation by UV light, thus creating a hotspot effect but only after a first mutational hit. In agreement, atypical TERT p mutations co-occur with canonical driver mutations in large cancer cohorts and arise subclonally specifically on the TERT p driver mutant chromosome homolog of melanoma cells treated with UV light in vitro. Our study gives an in-depth view of TERT p mutations in cancer and provides a mechanistic explanation for atypical TERT p mutations. Mutations at two key positions in the TERT promoter are frequent drivers of cancer, but the role of rare, atypical mutations in this region remains unclear. Here, the authors demonstrate that atypical TERT promoter mutations develop after canonical ones due to locally elevated UV-induced DNA damage.

Transcription factor A (TFAM) functions as a DNA packaging factor in mammalian mitochondria. TFAM also binds sequence-specifically to sites immediately upstream of mitochondrial promoters, but there are conflicting data regarding its role as a core component of the mitochondrial transcription machinery. We here demonstrate that TFAM is required for transcription in mitochondrial extracts as well as in a reconstituted in vitro transcription system. The absolute requirement of TFAM can be relaxed by conditions that allow DNA breathing, i.e., low salt concentrations or negatively supercoiled DNA templates. The situation is thus very similar to that described in nuclear RNA polymerase II-dependent transcription, in which the free energy of supercoiling can circumvent the need for a subset of basal transcription factors at specific promoters. In agreement with these observations, we demonstrate that TFAM has the capacity to induce negative supercoils in DNA, and, using the recently developed nucleobase analog FRET-pair tC ᴼ–tC ₙᵢₜᵣₒ, we find that TFAM distorts significantly the DNA structure. Our findings differ from recent observations reporting that TFAM is not a core component of the mitochondrial transcription machinery. Instead, our findings support a model in which TFAM is absolutely required to recruit the transcription machinery during initiation of transcription.