Mammalian mitochondrial DNA (mtDNA) encodes 13 proteins that are essential for the function of the oxidative phosphorylation system, which is composed of four respiratory-chain complexes and adenosine triphosphate (ATP) synthase. Remarkably, the maintenance and expression of mtDNA depend on the mitochondrial import of hundreds of nuclear-encoded proteins that control genome maintenance, replication, transcription, RNA maturation, and mitochondrial translation. The importance of this complex regulatory system is underscored by the identification of numerous mutations of nuclear genes that impair mtDNA maintenance and expression at different levels, causing human mitochondrial diseases with pleiotropic clinical manifestations. The basic scientific understanding of the mechanisms controlling mtDNA function has progressed considerably during the past few years, thanks to advances in biochemistry, genetics, and structural biology. The challenges for the future will be to understand how mtDNA maintenance and expression are regulated and to what extent direct intramitochondrial cross talk between different processes, such as transcription and translation, is important.

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 mitochondrial transcription machinery synthesizes the RNA primers required for initiation of leading-strand DNA synthesis in mammalian mitochondria. RNA primers are also required for initiation of lagging-strand DNA synthesis, but the responsible enzyme has so far remained elusive. Here, we present a series of observations that suggests that mitochondrial RNA polymerase (POLRMT) can act as lagging-strand primase in mammalian cells. POLRMT is highly processive on double-stranded DNA, but synthesizes RNA primers with a length of 25 to 75 nt on a single-stranded template. The short RNA primers synthesized by POLRMT are used by the mitochondrial DNA polymerase γ to initiate DNA synthesis in vitro . Addition of mitochondrial single-stranded DNA binding protein (mtSSB) reduces overall levels of primer synthesis, but stimulates primer-dependent DNA synthesis. Furthermore, when combined, POLRMT, DNA polymerase γ, the DNA helicase TWINKLE, and mtSSB are capable of simultaneous leading- and lagging-strand DNA synthesis in vitro . Based on our observations, we suggest that POLRMT is the lagging-strand primase in mammalian mitochondria.

Mammalian mitochondria contain multiple copies of a circular, double-stranded DNA genome and a dedicated DNA replication machinery is required for its maintenance. Many disease-causing mutations affect mitochondrial replication factors and a detailed understanding of the replication process may help to explain the pathogenic mechanisms underlying a number of mitochondrial diseases. We here give a brief overview of DNA replication in mammalian mitochondria, describing our current understanding of this process and some unanswered questions remaining.

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.