In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication

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.
Author Summary Mitochondria are cytoplasmatic organelles that produce most of the adenosine triphosphate (ATP) used by the cell as a source of chemical energy. A subset of proteins required for ATP production is encoded by a distinct mitochondrial DNA genome (mtDNA). Proper maintenance of mtDNA is essential, since mutations or depletion of this circular molecule may lead to a number of different diseases and also contribute to normal ageing. We are interested in the molecular mechanisms that ensure correct replication and propagation of mtDNA. Even if many of the responsible enzymes have been identified, there is still a debate within our scientific field regarding the exact mode of mtDNA replication. We have here used a combination of in vitro biochemistry and in vivo protein-DNA interaction characterization to address this question. Our findings demonstrate that the mitochondrial single-stranded DNA-binding protein (mtSSB) restricts initiation of mtDNA replication to a specific origin of replication. By characterizing how mtSSB interacts with the two strands of mtDNA in vivo, we are able to directly demonstrate the relevance of one proposed mode of mitochondrial DNA replication and at the same time seriously question the validity of other, alternative modes that have been proposed over the years.