Mechanism of mammalian mitochondrial DNA replication: import of mitochondrial transcription factor A into isolated mitochondria stimulates 7S DNA synthesis

The light strand promoter of mammalian mitochondrial DNA gives rise to a primary transcript, but also to the RNA primer necessary for initiation of replication and 7S DNA synthesis as well as 7S RNA. Here we have studied the turnover of 7S DNA in isolated rat liver mitochondria and whether import of mitochondrial transcription factor A (mtTFA), which is necessary for transcription initiation, increases its rate of synthesis. 7S DNA was present as two species, probably due to two different sites of RNA–DNA transition. Time course and pulse–chase experiments showed that the half-life of this DNA is ∼45 min. Import of mtTFA, produced in vitro, into the mitochondrial matrix in stoichiometric amounts significantly increased the rate of 7S DNA formation. We conclude that isolated rat liver mitochondria faithfully synthesize and degrade 7S DNA and that increased matrix levels of mtTFA are sufficient to increase its rate of synthesis, strongly supporting the hypothesis that this process is transcription primed.