Defects of mitochondrial DNA (mtDNA) maintenance have recently been associated with inherited neurodegenerative and muscle diseases and the aging process. Twinkle is a nuclear-encoded mtDNA helicase, dominant mutations of which cause adult-onset progressive external ophthalmoplegia (PEO) with multiple mtDNA deletions. We have generated transgenic mice expressing mouse Twinkle with PEO patient mutations. Multiple mtDNA deletions accumulate in the tissues of these mice, resulting in progressive respiratory dysfunction and chronic late-onset mitochondrial disease starting at 1 year of age. The muscles of the mice faithfully replicate all of the key histological, genetic, and biochemical features of PEO patients. Furthermore, the mice have progressive deficiency of cytochrome c oxidase in distinct neuronal populations. These “deletor” mice do not, however, show premature aging, indicating that subtle accumulation of mtDNA deletions and progressive respiratory chain dysfunction are not sufficient to accelerate aging. This model is a valuable tool for therapy development and testing for adult-onset mitochondrial disorders.

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.