Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element.

The majority of mitochondrial DNA replication events are terminated prematurely. The nascent DNA remains stably associated with the template, forming a triple-stranded displacement loop (D-loop) structure. However, the function of the D-loop region of the mitochondrial genome remains poorly understood. Using a comparative genomics approach we here identify two closely related 15 nt sequence motifs of the D-loop, strongly conserved among vertebrates. One motif is at the D-loop 5'-end and is part of the conserved sequence block 1 (CSB1). The other motif, here denoted coreTAS, is at the D-loop 3'-end. Both these sequences may prevent transcription across the D-loop region, since light and heavy strand transcription is terminated at CSB1 and coreTAS, respectively. Interestingly, the replication of the nascent D-loop strand, occurring in a direction opposite to that of heavy strand transcription, is also terminated at coreTAS, suggesting that coreTAS is involved in termination of both transcription and replication. Finally, we demonstrate that the loading of the helicase TWINKLE at coreTAS is reversible, implying that this site is a crucial component of a switch between D-loop formation and full-length mitochondrial DNA replication.

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.

MTERF1 binds mtDNA to prevent transcriptional interference at the light-strand promoter but is dispensable for rRNA gene transcription regulation.

Mitochondrial transcription termination factor 1, MTERF1, has been reported to couple rRNA gene transcription initiation with termination and is therefore thought to be a key regulator of mammalian mitochondrial ribosome biogenesis. The prevailing model is based on a series of observations published over the last two decades, but no in vivo evidence exists to show that MTERF1 regulates transcription of the heavy-strand region of mtDNA containing the rRNA genes. Here, we demonstrate that knockout of Mterf1 in mice has no effect on mitochondrial rRNA levels or mitochondrial translation. Instead, loss of Mterf1 influences transcription initiation at the light-strand promoter, resulting in a decrease of de novo transcription manifested as reduced 7S RNA levels. Based on these observations, we suggest that MTERF1 does not regulate heavy-strand transcription, but rather acts to block transcription on the opposite strand of mtDNA to prevent transcription interference at the light-strand promoter.

Subnormal levels of POLγA cause inefficient initiation of light-strand DNA synthesis and lead to mitochondrial DNA deletions and progressive external ophthalmoplegia [corrected].

The POLG1 gene encodes the catalytic subunit of mitochondrial DNA (mtDNA) polymerase γ (POLγ). We here describe a sibling pair with adult-onset progressive external ophthalmoplegia, cognitive impairment and mitochondrial myopathy characterized by DNA depletion and multiple mtDNA deletions. The phenotype is due to compound heterozygous POLG1 mutations, T914P and the intron mutation c.3104 + 3A > T. The mutant genes produce POLγ isoforms with heterozygous phenotypes that fail to synthesize longer DNA products in vitro. However, exon skipping in the c.3104 + 3A > T mutant is not complete, and the presence of low levels of wild-type POLγ explains patient survival. To better understand the underlying pathogenic mechanisms, we characterized the effects of POLγ depletion in vitro and found that leading-strand DNA synthesis is relatively undisturbed. In contrast, initiation of lagging-strand DNA synthesis is ineffective at lower POLγ concentrations that uncouples leading strand from lagging-strand DNA synthesis. In vivo, this effect leads to prolonged exposure of the heavy strand in its single-stranded conformation that in turn can cause the mtDNA deletions observed in our patients. Our findings, thus, suggest a molecular mechanism explaining how POLγ mutations can cause mtDNA deletions in vivo.

TWINKLE is an essential mitochondrial helicase required for synthesis of nascent D-loop strands and complete mtDNA replication.

Replication of the mammalian mitochondrial DNA (mtDNA) is dependent on the minimal replisome, consisting of the heterotrimeric mtDNA polymerase (POLG), the hexameric DNA helicase TWINKLE and the tetrameric single-stranded DNA-binding protein (mtSSB). TWINKLE has been shown to unwind DNA during the replication process and many disease-causing mutations have been mapped to its gene. Patients carrying Twinkle mutations develop multiple deletions of mtDNA, deficient respiratory chain function and neuromuscular symptoms. Despite its importance in human disease, it has been unclear whether TWINKLE is the only replicative DNA helicase in mammalian mitochondria. Furthermore, a substantial portion of mtDNA replication events is prematurely terminated at the end of mitochondrial control region (D-loop) and it is unknown whether TWINKLE also has a role in this abortive replication. Here, we present a conditional mouse knockout for Twinkle and demonstrate that TWINKLE is essential for mouse embryonic development and thus is the only replicative DNA helicase in mammalian mitochondria. Conditional knockout of Twinkle results in severe and rapid mtDNA depletion in heart and skeletal muscle. No replication intermediates or deleted mtDNA molecules are observed after Twinkle knockout, suggesting that TWINKLE once loaded is very processive. We also demonstrate that TWINKLE is essential for nascent H-strand synthesis in the D-loop, thus showing that there is no separate DNA helicase responsible for replication of this region. Our data thus suggest that the relative levels of abortive D-loop synthesis versus complete mtDNA replication are regulated and may provide a mechanism to control progression to complete mtDNA replication.

The mitochondrial DNA helicase TWINKLE can assemble on a closed circular template and support initiation of DNA synthesis.

Mitochondrial DNA replication is performed by a simple machinery, containing the TWINKLE DNA helicase, a single-stranded DNA-binding protein, and the mitochondrial DNA polymerase γ. In addition, mitochondrial RNA polymerase is required for primer formation at the origins of DNA replication. TWINKLE adopts a hexameric ring-shaped structure that must load on the closed circular mtDNA genome. In other systems, a specialized helicase loader often facilitates helicase loading. We here demonstrate that TWINKLE can function without a specialized loader. We also show that the mitochondrial replication machinery can assemble on a closed circular DNA template and efficiently elongate a DNA primer in a manner that closely resembles initiation of mtDNA synthesis in vivo.

Mitochondrial RNA polymerase is needed for activation of the origin of light-strand DNA replication.

Mitochondrial DNA is replicated by a unique enzymatic machinery, which is distinct from the replication apparatus used for copying the nuclear genome. We examine here the mechanisms of origin-specific initiation of lagging-strand DNA synthesis in human mitochondria. We demonstrate that the mitochondrial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the light-strand origin of DNA replication (OriL). Using only purified POLRMT and DNA replication factors, we can faithfully reconstitute OriL-dependent initiation in vitro. Leading-strand DNA synthesis is initiated from the heavy-strand origin of DNA replication and passes OriL. The single-stranded OriL is exposed and adopts a stem-loop structure. At this stage, POLRMT initiates primer synthesis from a poly-dT stretch in the single-stranded loop region. After about 25 nt, POLRMT is replaced by DNA polymerase gamma, and DNA synthesis commences. Our findings demonstrate that POLRMT can function as an origin-specific primase in mammalian mitochondria.