Abstract
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.
Highlights
In their inner membrane, mitochondria harbor the oxidative phosphorylation (OXPHOS) system, which generates adenosine triphosphate (ATP) needed to drive energetically unfavorable cellular reactions
Others have demonstrated that mitochondrial single-stranded DNA binding protein (mtSSB) is actively recruited to nucleoids during mitochondrial DNA genome (mtDNA) replication in vivo [34]
In agreement with early electron microscopy studies of rat mtDNA, our data demonstrate that mtSSB covers displaced single strands of replicative intermediates [36]
Summary
Mitochondria harbor the oxidative phosphorylation (OXPHOS) system, which generates ATP needed to drive energetically unfavorable cellular reactions. Most OXPHOS components are encoded in the nuclear genome, but genes for 13 essential subunits are encoded by a separate mitochondrial DNA genome (mtDNA). The two strands of the mitochondrial genome are known as the heavy strand (H-strand) and light strand (L-strand), owing to a strand bias in guanine and thymine base content. The genome is replicated by a molecular machinery that is distinct from the nuclear replication apparatus. The core components of this machinery are related to their phage T7 counterparts, including the catalytic subunit of DNA polymerase c (POLcA), the DNA helicase TWINKLE, and the mitochondrial RNA polymerase (POLRMT), which synthesizes
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