| 1. |
- Connor, T. M., et al.
(författare)
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Mutations in mitochondrial DNA causing tubulointerstitial kidney disease
- 2017
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Ingår i: Plos Genetics. - : Public Library of Science (PLoS). - 1553-7404. ; 13:3
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Tidskriftsartikel (refereegranskat)abstract
- Tubulointerstitial kidney disease is an important cause of progressive renal failure whose aetiology is incompletely understood. We analysed a large pedigree with maternally inherited tubulointerstitial kidney disease and identified a homoplasmic substitution in the control region of the mitochondrial genome (m.547A> T). While mutations in mtDNA coding sequence are a well recognised cause of disease affecting multiple organs, mutations in the control region have never been shown to cause disease. Strikingly, our patients did not have classical features of mitochondrial disease. Patient fibroblasts showed reduced levels of mitochondrial tRNA(Phe), tRNA(Leu1) and reduced mitochondrial protein translation and respiration. Mitochondrial transfer demonstrated mitochondrial transmission of the defect and in vitro assays showed reduced activity of the heavy strand promoter. We also identified further kindreds with the same phenotype carrying a homoplasmic mutation in mitochondrial tRNA Phe (m.616T> C). Thus mutations in mitochondrial DNA can cause maternally inherited renal disease, likely mediated through reduced function of mitochondrial tRNA(Phe)
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| 2. |
- Kuhl, I., et al.
(författare)
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POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA
- 2016
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 2:8
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondria are vital in providing cellular energy via their oxidative phosphorylation system, which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Transcription of the circular mammalian mtDNA depends on a single mitochondrial RNA polymerase (POLRMT). Although the transcription initiation process is well understood, it is debated whether POLRMT also serves as the primase for the initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in the heart results in severe mitochondrial dysfunction causing dilated cardiomyopathy in young mice. We further studied the molecular consequences of different expression levels of POLRMT and found that POLRMT is essential for primer synthesis to initiate mtDNA replication in vivo. Furthermore, transcription initiation for primer formation has priority over gene expression. Surprisingly, mitochondrial transcription factor A (TFAM) exists in an mtDNA-free pool in the Polrmt knockout mice. TFAM levels remain unchanged despite strong mtDNA depletion, and TFAM is thus protected from degradation of the AAA(+) Lon protease in the absence of POLRMT. Last, we report that mitochondrial transcription elongation factor may compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role of this factor in transcription. In conclusion, we present in vivo evidence that POLRMT has a key regulatory role in the replication of mammalian mtDNA and is part of a transcriptional mechanism that provides a switch between primer formation for mtDNA replication and mitochondrial gene expression.
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| 3. |
- Kukat, C., et al.
(författare)
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Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid
- 2015
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 112:36, s. 11288-11293
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Tidskriftsartikel (refereegranskat)abstract
- Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, super-resolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.
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| 4. |
- Posse, Viktor, et al.
(författare)
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Eukaryotic DNA replication with purified budding yeast proteins
- 2021
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Ingår i: The DNA replication-repair interface. - : Elsevier. - 9780323907330 ; , s. 1-33
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Bokkapitel (refereegranskat)abstract
- The in vitro reconstitution of origin firing was a key step toward the biochemical reconstitution of eukaryotic DNA replication in budding yeast. Today the basic replication assay involves proteins purified from 24 separate protocols that have evolved since their first publication, and as a result, the efficiency and reliability of the in vitro replication system has improved. Here we will present protocols for all 24 purifications together with a general protocol for the in vitro replication assay and some tips for troubleshooting problems with the assay.
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| 5. |
- Posse, Viktor, et al.
(författare)
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Human Mitochondrial Transcription Factor B2 Is Required for Promoter Melting during Initiation of Transcription
- 2017
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 292:7, s. 2637-2645
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Tidskriftsartikel (refereegranskat)abstract
- The mitochondrial transcription initiation machinery in humans consists of three proteins: the RNA polymerase (POLRMT) and two accessory factors, transcription factors A and B2 (TFAM and TFB2M, respectively). This machinery is required for the expression of mitochondrial DNA and the biogenesis of the oxidative phosphorylation system. Previous experiments suggested that TFB2M is required for promoter melting, but conclusive experimental proof for this effect has not been presented. Moreover, the role of TFB2M in promoter unwinding has not been discriminated from that of TFAM. Here we used potassium permanganate footprinting, DNase I footprinting, and in vitro transcription from the mitochondrial light-strand promoter to study the role of TFB2M in transcription initiation. We demonstrate that a complex composed of TFAM and POLRMT was readily formed at the promoter but alone was insufficient for promoter melting, which only occurred when TFB2M joined the complex. We also show that mismatch bubble templates could circumvent the requirement of TFB2M, but TFAM was still required for efficient initiation. Our findings support a model in which TFAM first recruits POLRMT to the promoter, followed by TFB2M binding and induction of promoter melting.
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| 6. |
- Posse, Viktor, et al.
(författare)
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RNase H1 directs origin-specific initiation of DNA replication in human mitochondria
- 2019
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Ingår i: Plos Genetics. - : Public Library of Science (PLoS). - 1553-7404. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after 120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3-ends that can be used by DNA polymerase to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggest a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria. Author summary Human mitochondria contain a double-stranded DNA genome that codes for key components of the oxidative phosphorylation system. The mitochondrial DNA (mtDNA) is replicated by a replication machinery distinct from that operating in the nucleus and mutations affecting individual replication factors have been associated with an array of rare, human diseases. In the present work, we demonstrate that RNase H1 directs origin-specific initiation of DNA replication in human mitochondria and that disease-causing mutations may impair this process. A unique feature of mtDNA replication is that primers required for initiation of leading-strand DNA replication are produced by the mitochondrial transcription machinery. A substantial fraction of all transcription events is prematurely terminated about 120 nucleotides downstream of the promoter and the RNA remains firmly associated with the genome, forming R-loops. Interestingly, the free 3-end of these R-loops cannot directly prime initiation of DNA synthesis, but must first be processed by RNase H1. The process is stimulated by the mitochondrial single-stranded DNA binding protein and faithfully reconstitutes replication events mapped in vivo. In combination with mapping of replication events in fibroblasts derived from patients with mutations in RNASEH1, our findings point to a possible model for replication initiation in human mitochondria similar to that previously described in the E. coli plasmid, ColE1.
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| 7. |
- Posse, Viktor, et al.
(författare)
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TEFM is a potent stimulator of mitochondrial transcription elongation in vitro
- 2015
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 43:5, s. 2615-2624
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Tidskriftsartikel (refereegranskat)abstract
- A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest. In the current work, we have isolated active TEFM in recombinant form and used a reconstituted in vitro transcription system to characterize its activities. We show that TEFM strongly promotes POLRMT processivity as it dramatically stimulates the formation of longer transcripts. TEFM also abolishes premature transcription termination at conserved sequence block II, an event that has been linked to primer formation during initiation of mtDNA synthesis. We show that POLRMT pauses at a wide range of sites in a given DNA sequence. In the absence of TEFM, this leads to termination; however, the presence of TEFM abolishes this effect and aids POLRMT in continuation of transcription. Further, we show that TEFM substantially increases the POLRMT affinity to an elongation-like DNA: RNA template. In combination with previously published in vivo observations, our data establish TEFM as an essential component of the mitochondrial transcription machinery.
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| 8. |
- Posse, Viktor, et al.
(författare)
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The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
- 2014
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 42:6, s. 3638-3647
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Tidskriftsartikel (refereegranskat)abstract
- Mammalian mitochondrial transcription is executed by a single subunit mitochondrial RNA polymerase (Polrmt) and its two accessory factors, mitochondrial transcription factors A and B2 (Tfam and Tfb2m). Polrmt is structurally related to single-subunit phage RNA polymerases, but it also contains a unique N-terminal extension (NTE) of unknown function. We here demonstrate that the NTE functions together with Tfam to ensure promoter-specific transcription. When the NTE is deleted, Polrmt can initiate transcription in the absence of Tfam, both from promoters and non-specific DNA sequences. Additionally, when in presence of Tfam and a mitochondrial promoter, the NTE-deleted mutant has an even higher transcription activity than wild-type polymerase, indicating that the NTE functions as an inhibitory domain. Our studies lead to a model according to which Tfam specifically recruits wild-type Polrmt to promoter sequences, relieving the inhibitory effect of the NTE, as a first step in transcription initiation. In the second step, Tfb2m is recruited into the complex and transcription is initiated.
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| 9. |
- Shi, Yonghong, et al.
(författare)
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Mitochondrial transcription termination factor 1 directs polar replication fork pausing
- 2016
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 44:12, s. 5732-5742
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Singh, Meenakshi, et al.
(författare)
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Ribonucleotides embedded in template DNA impair mitochondrial RNA polymerase progression
- 2022
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 50:2, s. 989-999
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Tidskriftsartikel (refereegranskat)abstract
- Human mitochondria lack ribonucleotide excision repair pathways, causing misincorporated ribonucleotides (rNMPs) to remain embedded in the mitochondrial genome. Previous studies have demonstrated that human mitochondrial DNA polymerase γ can bypass a single rNMP, but that longer stretches of rNMPs present an obstacle to mitochondrial DNA replication. Whether embedded rNMPs also affect mitochondrial transcription has not been addressed. Here we demonstrate that mitochondrial RNA polymerase elongation activity is affected by a single, embedded rNMP in the template strand. The effect is aggravated at stretches with two or more consecutive rNMPs in a row and cannot be overcome by addition of the mitochondrial transcription elongation factor TEFM. Our findings lead us to suggest that impaired transcription may be of functional relevance in genetic disorders associated with imbalanced nucleotide pools and higher levels of embedded rNMPs. © The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.
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