| 1. |
- Al-Behadili, Ali, et al.
(author)
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A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
- 2018
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In: Nucleic acids research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 46:18, s. 9471-9483
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Journal article (peer-reviewed)abstract
- 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.
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| 2. |
- Basu, Swaraj, et al.
(author)
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Accurate mapping of mitochondrial DNA deletions and duplications using deep sequencing
- 2020
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In: PLoS Genetics. - : Public Library of Science (PLoS). - 1553-7404. ; 16:12
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Journal article (peer-reviewed)abstract
- Deletions and duplications in mitochondrial DNA (mtDNA) cause mitochondrial disease and accumulate in conditions such as cancer and age-related disorders, but validated high-throughput methodology that can readily detect and discriminate between these two types of events is lacking. Here we establish a computational method, MitoSAlt, for accurate identification, quantification and visualization of mtDNA deletions and duplications from genomic sequencing data. Our method was tested on simulated sequencing reads and human patient samples with single deletions and duplications to verify its accuracy. Application to mouse models of mtDNA maintenance disease demonstrated the ability to detect deletions and duplications even at low levels of heteroplasmy. Author summary Deletions in the mitochondrial genome cause a wide variety of rare disorders, but are also linked to more common conditions such as neurodegeneration, diabetes type 2, and the normal ageing process. There is also a growing awareness that mtDNA duplications, which are also relevant for human disease, may be more common than previously thought. Despite their clinical importance, our current knowledge about the abundance, characteristics and diversity of mtDNA deletions and duplications is fragmented, and based to large extent on a limited view provided by traditional low-throughput analyses. Here, we describe a bioinformatics method, MitoSAlt, that can accurately map and classify mtDNA deletions and duplications using high-throughput sequencing. Application of this methodology to mouse models of mitochondrial deficiencies revealed a large number of duplications, suggesting that these may previously have been underestimated.
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| 3. |
- Jemt, Elisabeth, et al.
(author)
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Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element
- 2015
-
In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 43:19, s. 9262-9275
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Journal article (peer-reviewed)abstract
- 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.
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| 4. |
- Macao, Bertil, 1969, et al.
(author)
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The exonuclease activity of DNA polymerase gamma is required for ligation during mitochondrial DNA replication
- 2015
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 6
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Journal article (peer-reviewed)abstract
- Mitochondrial DNA (mtDNA) polymerase gamma (POL gamma) harbours a 3'-5' exonuclease proofreading activity. Here we demonstrate that this activity is required for the creation of ligatable ends during mtDNA replication. Exonuclease-deficient POL gamma fails to pause on reaching a downstream 5'-end. Instead, the enzyme continues to polymerize into double-stranded DNA, creating an unligatable 5'-flap. Disease-associated mutations can both increase and decrease exonuclease activity and consequently impair DNA ligation. In mice, inactivation of the exonuclease activity causes an increase in mtDNA mutations and premature ageing phenotypes. These mutator mice also contain high levels of truncated, linear fragments of mtDNA. We demonstrate that the formation of these fragments is due to impaired ligation, causing nicks near the origin of heavy-strand DNA replication. In the subsequent round of replication, the nicks lead to double-strand breaks and linear fragment formation.
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| 5. |
- Matic, S., et al.
(author)
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Mice lacking the mitochondrial exonuclease MGME1 accumulate mtDNA deletions without developing progeria
- 2018
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9
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Journal article (peer-reviewed)abstract
- Replication of mammalian mitochondrial DNA (mtDNA) is an essential process that requires high fidelity and control at multiple levels to ensure proper mitochondrial function. Mutations in the mitochondrial genome maintenance exonuclease 1 (MGME1) gene were recently reported in mitochondrial disease patients. Here, to study disease pathophysiology, we generated Mgme1 knockout mice and report that homozygous knockouts develop depletion and multiple deletions of mtDNA. The mtDNA replication stalling phenotypes vary dramatically in different tissues of Mgme1 knockout mice. Mice with MGME1 deficiency accumulate a long linear subgenomic mtDNA species, similar to the one found in mtDNA mutator mice, but do not develop progeria. This finding resolves a long-standing debate by showing that point mutations of mtDNA are the main cause of progeria in mtDNA mutator mice. We also propose a role for MGME1 in the regulation of replication and transcription termination at the end of the control region of mtDNA.
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| 6. |
- Oldfors Hedberg, Carola, 1969, et al.
(author)
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Deep sequencing of mitochondrial DNA and characterization of a novel POLG mutation in a patient with arPEO.
- 2020
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In: Neurology. Genetics. - 2376-7839. ; 6:1
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Journal article (peer-reviewed)abstract
- To determine the pathogenicity of a novel POLG mutation in a man with late-onset autosomal recessive progressive external ophthalmoplegia using clinical, molecular, and biochemical analyses.A multipronged approach with detailed neurologic examinations, muscle biopsy analyses, molecular genetic studies, and in vitro biochemical characterization.The patient had slowly progressive bilateral ptosis and severely reduced horizontal and vertical gaze. Muscle biopsy showed slight variability in muscle fiber size, scattered ragged red fibers, and partial cytochrome c oxidase deficiency. Biallelic mutations were identified in the POLG gene encoding the catalytic A subunit of POLγ. One allele carried a novel mutation in the exonuclease domain (c.590T>C; p.F197S), and the other had a previously characterized null mutation in the polymerase domain (c.2740A>C; p.T914P). Biochemical characterization revealed that the novel F197S mutant protein had reduced exonuclease and DNA polymerase activities and confirmed that T914P was inactive. By deep sequencing of mitochondrial DNA (mtDNA) extracted from muscle, multiple large-scale rearrangements were mapped and quantified.The patient's phenotype was caused by biallelic POLG mutations, resulting in one inactive POLγA protein (T914P) and one with decreased polymerase and exonuclease activity (F197S). The reduction in polymerase activity explains the presence of multiple pathogenic large-scale deletions in the patient's mtDNA.
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| 7. |
- Persson, Örjan, 1974, et al.
(author)
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Copy-choice recombination during mitochondrial L-strand synthesis causes DNA deletions
- 2019
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10
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Journal article (peer-reviewed)abstract
- Mitochondrial DNA (mtDNA) deletions are associated with mitochondrial disease, and also accumulate during normal human ageing. The mechanisms underlying mtDNA deletions remain unknown although several models have been proposed. Here we use deep sequencing to characterize abundant mtDNA deletions in patients with mutations in mitochondrial DNA replication factors, and show that these have distinct directionality and repeat characteristics. Furthermore, we recreate the deletion formation process in vitro using only purified mitochondrial proteins and defined DNA templates. Based on our in vivo and in vitro findings, we conclude that mtDNA deletion formation involves copy-choice recombination during replication of the mtDNA light strand.
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| 8. |
- Posse, Viktor, et al.
(author)
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RNase H1 directs origin-specific initiation of DNA replication in human mitochondria
- 2019
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In: Plos Genetics. - : Public Library of Science (PLoS). - 1553-7404. ; 15:1
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Journal article (peer-reviewed)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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| 9. |
- Torrino, S., et al.
(author)
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UBTD1 is a mechano-regulator controlling cancer aggressiveness
- 2019
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In: Embo Reports. - : EMBO. - 1469-221X .- 1469-3178. ; 20:4
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Journal article (peer-reviewed)abstract
- Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with beta-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase beta-TrCP. Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.
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| 10. |
- Torrino, S., et al.
(author)
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UBTD1 regulates ceramide balance and endolysosomal positioning to coordinate EGFR signaling
- 2021
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In: Elife. - 2050-084X. ; 10
-
Journal article (peer-reviewed)abstract
- To adapt in an ever-changing environment, cells must integrate physical and chemical signals and translate them into biological meaningful information through complex signaling pathways. By combining lipidomic and proteomic approaches with functional analysis, we have shown that ubiquitin domain-containing protein 1 (UBTD1) plays a crucial role in both the epidermal growth factor receptor (EGFR) self-phosphorylation and its lysosomal degradation. On the one hand, by modulating the cellular level of ceramides through N-acylsphingosine amidohydrolase 1 (ASAH1) ubiquitination, UBTD1 controls the ligand-independent phosphorylation of EGFR. On the other hand, UBTD1, via the ubiquitination of Sequestosome 1 (SQSTM1/p62) by RNF26 and endolysosome positioning, participates in the lysosomal degradation of EGFR. The coordination of these two ubiquitin-dependent processes contributes to the control of the duration of the EGFR signal. Moreover, we showed that UBTD1 depletion exacerbates EGFR signaling and induces cell proliferation emphasizing a hitherto unknown function of UBTD1 in EGFR-driven human cell proliferation.
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| 11. |
- Uhler, Jay (Jennifer), et al.
(author)
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In Vitro Analysis of mtDNA Replication
- 2021
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In: Mitochondrial Gene Expression. - New York, NY : Springer US. - 1064-3745.
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Book chapter (peer-reviewed)abstract
- Human mitochondrial DNA is a small circular double-stranded molecule that is essential for cellular energy production. A specialized protein machinery replicates the mitochondrial genome, with DNA polymerase γ carrying out synthesis of both strands. According to the prevailing mitochondrial DNA replication model, the two strands are replicated asynchronously, with the leading heavy-strand initiating first, followed by the lagging light-strand. By using purified recombinant forms of the replication proteins and synthetic DNA templates, it is possible to reconstitute mitochondrial DNA replication in vitro. Here we provide details on how to differentially reconstitute replication of the leading- and lagging-strands. © 2021, Springer Science+Business Media, LLC, part of Springer Nature.
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| 12. |
- Uhler, Jay (Jennifer), et al.
(author)
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MGME1 processes flaps into ligatable nicks in concert with DNA polymerase gamma during mtDNA replication
- 2016
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 44:12, s. 5861-5871
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Journal article (peer-reviewed)abstract
- Recently, MGME1 was identified as a mitochondrial DNA nuclease with preference for single-stranded DNA (ssDNA) substrates. Loss-of-function mutations in patients lead to mitochondrial disease with DNA depletion, deletions, duplications and rearrangements. Here, we assess the biochemical role of MGME1 in the processing of flap intermediates during mitochondrial DNA replication using reconstituted systems. We show that MGME1 can cleave flaps to enable efficient ligation of newly replicated DNA strands in combination with POL gamma. MGME1 generates a pool of imprecisely cut products (short flaps, nicks and gaps) that are converted to ligatable nicks by POL gamma through extension or excision of the 3'-end strand. This is dependent on the 3'-5' exonuclease activity of POL gamma which limits strand displacement activity and enables POL gamma to back up to the nick by 3'-5' degradation. We also demonstrate that POL gamma-driven strand displacement is sufficient to generate DNA- but not RNA-flap substrates suitable for MGME1 cleavage and ligation during replication. Our findings have implications for RNA primer removal models, the 5'-end processing of nascent DNA at OriH, and DNA repair.
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| 13. |
- Uhler, Jay (Jennifer), et al.
(author)
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Primer removal during mammalian mitochondrial DNA replication
- 2015
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In: DNA Repair. - : Elsevier BV. - 1568-7864. ; 34, s. 28-38
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Research review (peer-reviewed)abstract
- The small circular mitochondrial genome in mammalian cells is replicated by a dedicated replisome, defects in which can cause mitochondrial disease in humans. A fundamental step in mitochondrial DNA (mtDNA) replication and maintenance is the removal of the RNA primers needed for replication initiation. The nucleases RNase H1, PEN1, DNA2, and MGME1 have been implicated in this process. Here we review the role of these nucleases in the light of primer removal pathways in mitochondria, highlight associations with disease, as well as consider the implications for mtDNA replication initiation. (C) 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license.
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| 14. |
- Uhler, Jay (Jennifer), et al.
(author)
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The UbL protein UBTD1 stably interacts with the UBE2D family of E2 ubiquitin conjugating enzymes
- 2014
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In: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 443:1, s. 7-12
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Journal article (peer-reviewed)abstract
- UBTD1 is a previously uncharacterized ubiquitin-like (UbL) domain containing protein with high homology to the mitochondrial Dc-UbP/UBTD2 protein. Here we show that UBTD1 and UBTD2 belong to a family of proteins that is conserved through evolution and found in metazoa, funghi, and plants. To gain further insight into the function of UBTD1, we screened for interacting proteins. In a yeast-2-hybrid (Y2H) screen, we identified several proteins involved in the ubiquitylation pathway, including the UBE2D family of E2 ubiquitin conjugating enzymes. An affinity capture screen for UBTD1 interacting proteins in whole cell extracts also identified members of the UBE2D family. Biochemical characterization of recombinant UBTD1 and UBE2D demonstrated that the two proteins form a stable, stoichiometric complex that can be purified to near homogeneity. We discuss the implications of these findings in light of the ubiquitin proteasome system (UPS). (C) 2013 Published by Elsevier Inc.
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| 15. |
- Wanrooij, Paulina, et al.
(author)
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A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop
- 2012
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:20, s. 10334-10344
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Journal article (peer-reviewed)abstract
- 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.
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| 16. |
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| 17. |
- Zhao, Y. B., et al.
(author)
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Identification of a G-quadruplex forming sequence in the promoter of UCP1
- 2018
-
In: Acta Biochimica Et Biophysica Sinica. - : China Science Publishing & Media Ltd.. - 1672-9145. ; 50:7, s. 718-722
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Journal article (peer-reviewed)abstract
- G-quadruplexes are higher-order nucleic acid structures formed in G-rich sequences in DNA or RNA. G-quadruplexes are distributed in many locations in the human genome, including promoter regions, and are viewed as promising therapeutic targets. Uncoupling protein-1 (UCP1) is a mitochondrial thermogenic gene critical for energy expenditure in the form of heat in the brown adipose tissue. UCP1 is only expressed during brown fat cell differentiation and is a candidate target for treating obesity. However, the regulation of UCP1 expression is not clear. We reported here that a G-quadruplex forming sequence exists in the promoter of UCP1. The 5,10,15,20-tetra(Nmethyl- 4-pyridyl) porphyrin (TMPyP4) enhanced cellular expression of UCP1 and destabilized the G-quadruplex formed by the sequence from the promoter of UCP1. Mutations in the G-quadruplex regulated the cellular activity of UCP1 promoter as evidenced by a UCP1-promoter luciferase assay. These results suggest that G-quadruplex structure is a potential target to regulate the expression of UCP1.
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