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.

The human mitochondrial transcription machinery generates the primers required for initiation of leading-strand DNA replication. According to one model, the 3′ end of the primer is defined by transcription termination at conserved sequence block II (CSB II) in the mitochondrial DNA control region. We here demonstrate that this site-specific termination event is caused by G-quadruplex structures formed in nascent RNA upon transcription of CSB II. We also demonstrate that a poly-dT stretch downstream of CSB II has a modest stimulatory effect on the termination efficiency. The mechanism is reminiscent of Rho-independent transcription termination in prokaryotes, with the exception that a G-quadruplex structure replaces the hairpin loop formed in bacterial mRNA during transcription of terminator sequences.

In human mitochondria the transcription machinery generates the RNA primers needed for initiation of DNA replication. A critical feature of the leading-strand origin of mitochondrial DNA replication is a CG-rich element denoted conserved sequence block II (CSB II). During transcription of CSB II, a G-quadruplex structure forms in the nascent RNA, which stimulates transcription termination and primer formation. Previous studies have shown that the newly synthesized primers form a stable and persistent RNA–DNA hybrid, a R-loop, near the leading-strand origin of DNA replication. We here demonstrate that the unusual behavior of the RNA primer is explained by the formation of a stable G-quadruplex structure, involving the CSB II region in both the nascent RNA and the non-template DNA strand. Based on our data, we suggest that G-quadruplex formation between nascent RNA and the non-template DNA strand may be a regulated event, which decides the fate of RNA primers and ultimately the rate of initiation of DNA synthesis in human mitochondria.

The mechanisms of mitochondrial DNA replication have been hotly debated for a decade. The strand‐displacement model states that lagging‐strand DNA synthesis is initiated from the origin of light‐strand DNA replication (OriL), whereas the strand‐coupled model implies that OriL is dispensable. Mammalian mitochondria cannot be transfected and the requirements of OriL in vivo have therefore not been addressed. We here use in vivo saturation mutagenesis to demonstrate that OriL is essential for mtDNA maintenance in the mouse. Biochemical and bioinformatic analyses show that OriL is functionally conserved in vertebrates. Our findings strongly support the strand‐displacement model for mtDNA replication. There is a [Hot off the Press][1] (December 2012) associated with this Scientific Report. [1]: http://dx.doi.org/10.1038/embor.2012.175

Oxidative stress is capable of causing damage to various cellular constituents, including DNA. There is however limited knowledge on how oxidative stress influences mitochondrial DNA and its replication. Here, we have used purified mtDNA replication proteins, i.e. DNA polymerase γ holoenzyme, the mitochondrial single-stranded DNA binding protein mtSSB, the replicative helicase Twinkle and the proposed mitochondrial translesion synthesis polymerase PrimPol to study lesion bypass synthesis on oxidative damage-containing DNA templates. Our studies were carried out at dNTP levels representative of those prevailing either in cycling or in non-dividing cells. At dNTP concentrations that mimic those in cycling cells, the replication machinery showed substantial stalling at sites of damage and these problems were further exacerbated at the lower dNTP concentrations present in resting cells. PrimPol, the translesion synthesis polymerase identified inside mammalian mitochondria, did not promote mtDNA replication fork bypass of the damage. This argues against a conventional role for PrimPol as a mitochondrial translesion synthesis DNA polymerase for oxidative DNA damage; however, we show that Twinkle, the mtDNA replicative helicase, is able to stimulate PrimPol DNA synthesis in vitro, suggestive of an as yet unidentified role of PrimPol in mtDNA metabolism.

Mitochondria are central to numerous metabolic pathways whereby mitochondrial dysfunction has a profound impact and can manifest in disease. The consequences of mitochondrial dysfunction can be ameliorated by adaptive responses that rely on crosstalk from the mitochondria to the rest of the cell. Such mito-cellular signalling slows cell cycle progression in mitochondrial DNA–deficient (ρ⁰)Saccharomyces cerevisiaecells, but the initial trigger of the response has not been thoroughly studied. Here, we show that decreased mitochondrial membrane potential (ΔΨm) acts as the initial signal of mitochondrial stress that delays G1-to-S phase transition in both ρ⁰and control cells containing mtDNA. Accordingly, experimentally increasing ΔΨm was sufficient to restore timely cell cycle progression in ρ⁰cells. In contrast, cellular levels of oxidative stress did not correlate with the G1-to-S delay. Restored G1-to-S transition in ρ⁰cells with a recovered ΔΨm is likely attributable to larger cell size, whereas the timing of G1/S transcription remained delayed. The identification of ΔΨm as a regulator of cell cycle progression may have implications for disease states involving mitochondrial dysfunction.