Structure Casts Light on mtDNA Replication

In this issue, Lee et al. (2009) present a crystal structure of the human mitochondrial DNA polymerase (POLγ). The structure of this heterotrimeric enzyme lays a foundation for understanding how POLγ mutations cause human mitochondrial disease and why some antiviral nucleoside analogs cause cellular toxicity

A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop

In human mitochondria the transcription machinery generates the RNA primers needed for initiation of DNA replication. A critical feature of the leading-strand origin of mitochondrial DNA replication is a CG-rich element denoted conserved sequence block II (CSB II). During transcription of CSB II, a G-quadruplex structure forms in the nascent RNA, which stimulates transcription termination and primer formation. Previous studies have shown that the newly synthesized primers form a stable and persistent RNA-DNA hybrid, a R-loop, near the leading-strand origin of DNA replication. We here demonstrate that the unusual behavior of the RNA primer is explained by the formation of a stable G-quadruplex structure, involving the CSB II region in both the nascent RNA and the non-template DNA strand. Based on our data, we suggest that G-quadruplex formation between nascent RNA and the non-template DNA strand may be a regulated event, which decides the fate of RNA primers and ultimately the rate of initiation of DNA synthesis in human mitochondria

The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities

The TWINKLE protein is a hexameric DNA helicase required for replication of mitochondrial DNA. TWINKLE displays striking sequence similarity to the bacteriophage T7 gene 4 protein (gp4), which is a bi-functional primase-helicase required at the phage DNA replication fork. The N-terminal domain of human TWINKLE contains some of the characteristic sequence motifs found in the N-terminal primase domain of the T7 gp4, but other important motifs are missing. TWINKLE is not an active primase in vitro and the functional role of the N-terminal region has remained elusive. In this report, we demonstrate that the N-terminal part of TWINKLE is required for efficient binding to single-stranded DNA. Truncations of this region reduce DNA helicase activity and mitochondrial DNA replisome processivity. We also find that the gp4 and TWINKLE are functionally distinct. In contrast to the phage protein, TWINKLE binds to double-stranded DNA. Moreover, TWINKLE forms stable hexamers even in the absence of Mg2+ or NTPs, which suggests that an accessory protein, a helicase loader, is needed for loading of TWINKLE onto the circular mtDNA genome

Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system

Termination of transcription is a key process in the regulation of mitochondrial gene expression in animal cells. To investigate transcription termination in sea urchin mitochondria, we cloned the mitochondrial RNA polymerase (mtRNAP) of Paracentrotus lividus and used a recombinant form of the enzyme in a reconstituted transcription system, in the presence of the DNA-binding protein mtDBP. Cloning of mtRNAP was performed by a combination of PCR with degenerate primers and library screening. The enzyme contains 10 phage-like conserved motifs, two pentatricopeptide motifs and a serine-rich stretch. The protein expressed in insect cells supports transcription elongation in a promoter-independent assay. Addition of recombinant mtDBP caused arrest of the transcribing mtRNAP when the enzyme approached the mtDBP-binding site in the direction of transcription of mtDNA l-strand. When the polymerase encountered the protein-binding site in the opposite direction, termination occurred in a protein-independent manner, inside the mtDBP-binding site. Pulse-chase experiments show that mtDBP caused true transcription termination rather than pausing. These data indicate that mtDBP acts as polar termination factor and suggest that transcription termination in sea urchin mitochondria could take place by two alternative modes based on protein-mediated or sequence-dependent mechanisms

Molecular insights into mitochondrial DNA replication

Mitochondria are organelles found in eukaryotic cells. These organelles produce most of the adenosine triphosphate that cells use as a source of energy. Mitochondria contain their own genomic material, a circular DNA genome (mtDNA) that encodes subunits of the respiratory chain complexes and RNA components needed for mitochondrial translation. Many aspects of mtDNA replication are still not understood and in this thesis we address some of the molecular mechanisms of this process in mammalian cells. DNA synthesis cannot be initiated de novo, but requires a short RNA primer as a starting point. We here demonstrate that the mitochondrial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the origin of light strand DNA replication (OriL) in human mtDNA. Using purified POLRMT and the core factors of the mitochondrial replisome, we faithfully reconstitute OriLdependent initiation of replication in vitro. During origin activation, OriL is exposed in its single-stranded conformation and adopts a stem-loop structure. POLRMT initiates primer synthesis from a poly-dT stretch in the single-stranded loop region and after about 25 nt, POLRMT is replaced by the mitochondrial DNA polymerase ! (POL!) and DNA synthesis is initiated. Our findings also suggest that the mitochondrial single-stranded DNA binding protein directs origin-specific initiation by efficiently blocking unspecific initiation events in other regions of the mtDNA genome. To analyze the requirements of OriL in vivo, we have used saturation mutagenesis in the mouse combined with in vitro biochemistry and demonstrated that OriL is essential for mtDNA maintenance. OriL requires a stable stem-loop structure and a pyrimidine-rich sequence in the template strand for proper origin function. The OriL mechanism appears to be conserved, since bioinformatics analyses demonstrated the presence of OriL in the mtDNA of most vertebrates including birds. Our findings suggest that mtDNA replication may be performed by a common mechanism in all vertebrates and lend support to the strand-displacement model for mtDNA replication. A molecular understanding of the mitochondrial DNA replication machinery is also of medical importance. Today, more than 160 mutations in the gene encoding the catalytic subunit of POL! (POL!A) have been associated with human disease. One example is the Y955C mutation, which causes autosomal dominant progressive external ophthalmoplegia, a disorder characterized by the accumulation of multiple mtDNA deletions. The Y955C mutation decreases POL! processivity due to a decreased binding affinity for the incoming deoxyribonucleoside triphosphate. However, it is not clear why this biochemical defect leads to a dominant disease. We have used the reconstituted mammalian mtDNA replisome and studied functional consequences of the dominant Y955C mutation. Our study revealed that the POL!A:Y955C enzyme is prone to stalling at dATP insertion sites and instead enters a polymerase/exonuclease idling mode. The mutant POL!A:Y955C competes with wild-type POL!A for access to the primer template. However, once assembled in the replisome, the wild-type enzyme is no longer affected. Our data therefore provide a mechanism for the mtDNA replication phenotypes seen in patients harboring the Y955C mutation

Mitochondrial transcription termination factor 1 directs polar replication fork pausing

During replication of nuclear ribosomal DNA (rDNA), clashes with the transcription apparatus can cause replication fork collapse and genomic instability. To avoid this problem, a replication fork barrier protein is situated downstream of rDNA, there preventing replication in the direction opposite rDNA transcription. A potential candidate for a similar function in mitochondria is the mitochondrial transcription termination factor 1 (MTERF1, also denoted mTERF), which binds to a sequence just downstream of the ribosomal transcription unit. Previous studies have shown that MTERF1 prevents antisense transcription over the ribosomal RNA genes, a process which we here show to be independent of the transcription elongation factor TEFM. Importantly, we now demonstrate that MTERF1 arrests mitochondrial DNA (mtDNA) replication with distinct polarity. The effect is explained by the ability of MTERF1 to act as a directional contrahelicase, blocking mtDNA unwinding by the mitochondrial helicase TWINKLE. This conclusion is also supported by in vivo evidence that MTERF1 stimulates TWINKLE pausing. We conclude that MTERF1 can direct polar replication fork arrest in mammalian mitochondria.Peer reviewe

A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL.

The role of Ribonuclease H1 (RNase H1) during primer removal and ligation at the mitochondrial origin of light-strand DNA synthesis (OriL) is a key, yet poorly understood, step in mitochondrial DNA maintenance. Here, we reconstitute the replication cycle of L-strand synthesis in vitro using recombinant mitochondrial proteins and model OriL substrates. The process begins with initiation of DNA replication at OriL and ends with primer removal and ligation. We find that RNase H1 partially removes the primer, leaving behind the last one to three ribonucleotides. These 5'-end ribonucleotides disturb ligation, a conclusion which is supported by analysis of RNase H1-deficient patient cells. A second nuclease is therefore required to remove the last ribonucleotides and we demonstrate that Flap endonuclease 1 (FEN1) can execute this function in vitro. Removal of RNA primers at OriL thus depends on a two-nuclease model, which in addition to RNase H1 requires FEN1 or a FEN1-like activity. These findings define the role of RNase H1 at OriL and help to explain the pathogenic consequences of disease causing mutations in RNase H1

In Vitro-Reconstituted Nucleoids Can Block Mitochondrial DNA Replication and Transcription

SummaryThe mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000–10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication