| 1. |
- Boldinova, Elizaveta O., et al.
(författare)
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DNA Damage Tolerance by Eukaryotic DNA Polymerase and Primase PrimPol
- 2017
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 18:7
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Forskningsöversikt (refereegranskat)abstract
- PrimPol is a human deoxyribonucleic acid (DNA) polymerase that also possesses primase activity and is involved in DNA damage tolerance, the prevention of genome instability and mitochondrial DNA maintenance. In this review, we focus on recent advances in biochemical and crystallographic studies of PrimPol, as well as in identification of new protein-protein interaction partners. Furthermore, we discuss the possible functions of PrimPol in both the nucleus and the mitochondria.
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| 2. |
- Doimo, Mara, et al.
(författare)
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MtDNA replication, maintenance, and nucleoid organization
- 2020
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Ingår i: The human mitochondrial genome. - : Academic Press. - 9780128196564 ; , s. 3-33
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Bokkapitel (refereegranskat)abstract
- Part of the genetic information in human cells resides in the mitochondria. Faithful maintenance of mitochondrial deoxyribonucleic acid (mtDNA) is crucial for the oxidative phosphorylation system that produces the majority of the cellular ATP, and therefore to life. This chapter provides an introduction into the characteristics of human mtDNA and summarizes the processes and factors required for the replication and maintenance of this small but essential genome. We also describe the organization of mtDNA in specialized nucleoprotein structures called nucleoids. Where applicable, we refer to human disease states that are caused by defects in the described factors or processes.
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| 3. |
- Shi, Yonghong, et al.
(författare)
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Mammalian transcription factor A is a core component of the mitochondrial transcription machinery
- 2012
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Ingår i: Proceedings of the National Academy of Science of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 109:41, s. 16510-16515
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Tidskriftsartikel (refereegranskat)abstract
- Transcription factor A (TFAM) functions as a DNA packaging factor in mammalian mitochondria. TFAM also binds sequence-specifically to sites immediately upstream of mitochondrial promoters, but there are conflicting data regarding its role as a core component of the mitochondrial transcription machinery. We here demonstrate that TFAM is required for transcription in mitochondrial extracts as well as in a reconstituted in vitro transcription system. The absolute requirement of TFAM can be relaxed by conditions that allow DNA breathing, i.e., low salt concentrations or negatively supercoiled DNA templates. The situation is thus very similar to that described in nuclear RNA polymerase II-dependent transcription, in which the free energy of supercoiling can circumvent the need for a subset of basal transcription factors at specific promoters. In agreement with these observations, we demonstrate that TFAM has the capacity to induce negative supercoils in DNA, and, using the recently developed nucleobase analog FRET-pair tC(O)-tC(nitro), we find that TFAM distorts significantly the DNA structure. Our findings differ from recent observations reporting that TFAM is not a core component of the mitochondrial transcription machinery. Instead, our findings support a model in which TFAM is absolutely required to recruit the transcription machinery during initiation of transcription.
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| 4. |
- Stojkovic, Gorazd, et al.
(författare)
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Oxidative DNA damage stalls the human mitochondrial replisome
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Oxidative stress is capable of causing damage to various cellular constituents, including DNA. There is however limited knowledge on how oxidative stress influences mitochondrial DNA and its replication. Here, we have used purified mtDNA replication proteins, i.e. DNA polymerase. holoenzyme, the mitochondrial single-stranded DNA binding protein mtSSB, the replicative helicase Twinkle and the proposed mitochondrial translesion synthesis polymerase PrimPol to study lesion bypass synthesis on oxidative damage-containing DNA templates. Our studies were carried out at dNTP levels representative of those prevailing either in cycling or in non-dividing cells. At dNTP concentrations that mimic those in cycling cells, the replication machinery showed substantial stalling at sites of damage, and these problems were further exacerbated at the lower dNTP concentrations present in resting cells. PrimPol, the translesion synthesis polymerase identified inside mammalian mitochondria, did not promote mtDNA replication fork bypass of the damage. This argues against a conventional role for PrimPol as a mitochondrial translesion synthesis DNA polymerase for oxidative DNA damage; however, we show that Twinkle, the mtDNA replicative helicase, is able to stimulate PrimPol DNA synthesis in vitro, suggestive of an as yet unidentified role of PrimPol in mtDNA metabolism.
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| 5. |
- Wanrooij, Paulina H., et al.
(författare)
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Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired
- 2017
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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. ; 114:47, s. 12466-12471
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Tidskriftsartikel (refereegranskat)abstract
- Incorporation of ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove incorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs. Using a collection of yeast strains with altered dNTP pools, we discovered an inverse relationship between the concentration of individual dNTPs and the amount of the corresponding ribonucleotides incorporated in mitochondrial DNA, while in nuclear DNA the ribonucleotide pattern was only altered in the absence of ribonucleotide excision repair. Our analysis uncovers major differences in ribonucleotide repair between the two genomes and provides concrete evidence that yeast mitochondria lack mechanisms for removal of ribonucleotides incorporated by the mtDNA polymerase. Furthermore, as cytosolic dNTP pool imbalances were transmitted equally well into the nucleus and the mitochondria, our results support a view of the cytosolic and mitochondrial dNTP pools in frequent exchange. © 2017, National Academy of Sciences. All rights reserved.
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| 6. |
- Wanrooij, Sjoerd, et al.
(författare)
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In vivo mutagenesis reveals that OriL is essential for mitochondrial DNA replication.
- 2012
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Ingår i: EMBO reports. - : EMBO. - 1469-3178 .- 1469-221X. ; 13:12, s. 1130-7
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Tidskriftsartikel (refereegranskat)abstract
- The mechanisms of mitochondrial DNA replication have been hotly debated for a decade. The strand-displacement model states that lagging-strand DNA synthesis is initiated from the origin of light-strand DNA replication (OriL), whereas the strand-coupled model implies that OriL is dispensable. Mammalian mitochondria cannot be transfected and the requirements of OriL in vivo have therefore not been addressed. We here use in vivo saturation mutagenesis to demonstrate that OriL is essential for mtDNA maintenance in the mouse. Biochemical and bioinformatic analyses show that OriL is functionally conserved in vertebrates. Our findings strongly support the strand-displacement model for mtDNA replication.
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| 7. |
- Breidenstein, Annika, et al.
(författare)
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PrgE: an OB-fold protein from plasmid pCF10 with striking differences to prototypical bacterial SSBs
- 2024
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Ingår i: Life Science Alliance. - : Life Science Alliance. - 2575-1077. ; 7:8
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Tidskriftsartikel (refereegranskat)abstract
- A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded type IV secretion systems (T4SSs). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many, their function remains unclear. Here, we characterize PrgE, a proposed SSB from the Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very unusual DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string supported by its N-terminal tail. In vitro studies highlight the plasticity of PrgE oligomerization and confirm the importance of the N-terminus. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from eukaryotic SSBs.
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| 8. |
- Carvalho, Gustavo, et al.
(författare)
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Mitochondrial DNA Instability in Mammalian Cells
- 2022
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Ingår i: Antioxidants and Redox Signaling. - : Mary Ann Liebert. - 1523-0864 .- 1557-7716. ; 36:13-15, s. 885-905
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Forskningsöversikt (refereegranskat)abstract
- Significance: The small, multicopy mitochondrial genome (mitochondrial DNA [mtDNA]) is essential for efficient energy production, as alterations in its coding information or a decrease in its copy number disrupt mitochondrial ATP synthesis. However, the mitochondrial replication machinery encounters numerous challenges that may limit its ability to duplicate this important genome and that jeopardize mtDNA stability, including various lesions in the DNA template, topological stress, and an insufficient nucleotide supply.Recent Advances: An ever-growing array of DNA repair or maintenance factors are being reported to localize to the mitochondria. We review current knowledge regarding the mitochondrial factors that may contribute to the tolerance or repair of various types of changes in the mitochondrial genome, such as base damage, incorporated ribonucleotides, and strand breaks. We also discuss the newly discovered link between mtDNA instability and activation of the innate immune response.Critical Issues: By which mechanisms do mitochondria respond to challenges that threaten mtDNA maintenance? What types of mtDNA damage are repaired, and when are the affected molecules degraded instead? And, finally, which forms of mtDNA instability trigger an immune response, and how?Future Directions: Further work is required to understand the contribution of the DNA repair and damage-tolerance factors present in the mitochondrial compartment, as well as the balance between mtDNA repair and degradation. Finally, efforts to understand the events underlying mtDNA release into the cytosol are warranted. Pursuing these and many related avenues can improve our understanding of what goes wrong in mitochondrial disease.
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| 9. |
- Gorospe, Choco Michael, et al.
(författare)
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Determination of the ribonucleotide content of mtDNA using alkaline gels
- 2023
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Ingår i: Mitochondrial DNA. - New York : Humana Press. - 9781071629215 - 9781071629222 ; , s. 293-314
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Bokkapitel (refereegranskat)abstract
- Impaired mitochondrial DNA (mtDNA) maintenance, due to, e.g., defects in the replication machinery or an insufficient dNTP supply, underlies a number of mitochondrial disorders. The normal process of mtDNA replication leads to the incorporation of multiple single ribonucleotides (rNMPs) per mtDNA molecule. Given that embedded rNMPs alter the stability and properties of the DNA, they may have consequences for mtDNA maintenance and thereby for mitochondrial disease. They also serve as a readout of the intramitochondrial NTP/dNTP ratios. In this chapter, we describe a method for the determination of mtDNA rNMP content using alkaline gel electrophoresis and Southern blotting. This procedure is suited for the analysis of mtDNA in total genomic DNA preparations as well as in purified form. Moreover, it can be performed using equipment found in most biomedical laboratories, allows the simultaneous analysis of 10-20 samples depending on the gel system employed, and can be modified for the analysis of other mtDNA modifications.
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| 10. |
- Gorospe, Choco Michael, et al.
(författare)
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Mitochondrial membrane potential acts as a retrograde signal to regulate cell cycle progression
- 2023
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Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 6:12
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondria are central to numerous metabolic pathways whereby mitochondrial dysfunction has a profound impact and can manifest in disease. The consequences of mitochondrial dysfunction can be ameliorated by adaptive responses that rely on crosstalk from the mitochondria to the rest of the cell. Such mito-cellular signalling slows cell cycle progression in mitochondrial DNA-deficient (ρ0) Saccharomyces cerevisiae cells, but the initial trigger of the response has not been thoroughly studied. Here, we show that decreased mitochondrial membrane potential (ΔΨm) acts as the initial signal of mitochondrial stress that delays G1-to-S phase transition in both ρ0 and control cells containing mtDNA. Accordingly, experimentally increasing ΔΨm was sufficient to restore timely cell cycle progression in ρ0 cells. In contrast, cellular levels of oxidative stress did not correlate with the G1-to-S delay. Restored G1-to-S transition in ρ0 cells with a recovered ΔΨm is likely attributable to larger cell size, whereas the timing of G1/S transcription remained delayed. The identification of ΔΨm as a regulator of cell cycle progression may have implications for disease states involving mitochondrial dysfunction.
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| 11. |
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| 12. |
- Nystrom, K., et al.
(författare)
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Inosine triphosphate pyrophosphatase enhances the effect of ribavirin on hepatitis C virus cell culture infection
- 2017
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Ingår i: Journal of Hepatology. - : ELSEVIER SCIENCE BV. - 0168-8278 .- 1600-0641. ; 66:1, s. S321-S321
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Tidskriftsartikel (refereegranskat)abstract
- Genetic variants of the inosine triphosphate pyrophosphatase gene (ITPA), resulting in decreased enzymatic activity of the corresponding enzyme, ITPase, are known to correlate with a decreased risk of ribavirin-induced anemia, but are also associated with an increased SVR in patients treated with peginterferon-alpha and ribavirin. As both ITPase and ribavirin are involved in the nucleotide salvage pathway and reduced risk of relapse after treatment of hepatitis C, we have investigated the effect of ITPase activity and ribavirin treatment of HCVcc infection of hepatocytes
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| 13. |
- Nyström, Kristina, et al.
(författare)
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Inosine triphosphate pyrophosphatase dephosphorylates ribavirin triphosphate and reduced enzymatic activity potentiates mutagenesis in hepatitis C virus
- 2018
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Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 92:19
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Tidskriftsartikel (refereegranskat)abstract
- A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (ITPA) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against ITPA or a negative-control siRNA in the presence or absence of ribavirin in an HCV culture system. Low ribavirin concentrations strikingly depleted intracellular GTP levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions in the HCV genome, with an ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies. IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular GTP and increased hepatitis C virus mutagenesis. In cell culture, reduced ITP pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP, similar to ITP, a naturally occurring substrate of ITPase, is dephosphorylated in vitro by ITPase.
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| 14. |
- Repolês, Bruno Marçal, et al.
(författare)
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The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method
- 2021
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Ingår i: Mitochondrion (Amsterdam. Print). - : Elsevier. - 1567-7249 .- 1872-8278. ; 61, s. 179-187
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Tidskriftsartikel (refereegranskat)abstract
- The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues.
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| 15. |
- Sawicka, Marta, et al.
(författare)
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The dimeric architecture of checkpoint kinases Mec1(ATR) and Tel1(ATM) reveal a common structural organization
- 2016
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Ingår i: Journal of Biological Chemistry. - : Elsevier. - 0021-9258 .- 1083-351X. ; 291:26, s. 13436-13447
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Tidskriftsartikel (refereegranskat)abstract
- The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form.
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| 16. |
- Tran, Phong, et al.
(författare)
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De novo dNTP production is essential for normal postnatal murine heart development
- 2019
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 394:44, s. 15889-15897
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Tidskriftsartikel (refereegranskat)abstract
- The building blocks of DNA, dNTPs, can be produced de novo or can be salvaged from deoxyribonucleosides. However, to what extent the absence of de novo dNTP production can be compensated for by the salvage pathway is unknown. Here, we eliminated de novo dNTP synthesis in the mouse heart and skeletal muscle by inactivating ribonucleotide reductase (RNR), a key enzyme for the de novo production of dNTPs, at embryonic day 13. All other tissues had normal de novo dNTP synthesis and theoretically could supply heart and skeletal muscle with deoxyribonucleosides needed for dNTP production by salvage. We observed that the dNTP and NTP pools in wild-type postnatal hearts are unexpectedly asymmetric, with unusually high dGTP and GTP levels compared with those in whole mouse embryos or murine cell cultures. We found that RNR inactivation in heart led to strongly decreased dGTP and increased dCTP, dTTP, and dATP pools; aberrant DNA replication; defective expression of muscle-specific proteins; progressive heart abnormalities; disturbance of the cardiac conduction system; and lethality between the second and fourth weeks after birth. We conclude that dNTP salvage cannot substitute for de novo dNTP synthesis in the heart and that cardiomyocytes and myocytes initiate DNA replication despite an inadequate dNTP supply. We discuss the possible reasons for the observed asymmetry in dNTP and NTP pools in wildtype hearts.
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| 17. |
- Wanrooij, Paulina, et al.
(författare)
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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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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:20, s. 10334-10344
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Tidskriftsartikel (refereegranskat)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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| 18. |
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| 19. |
- Wanrooij, Paulina H., et al.
(författare)
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Elimination of rNMPs from mitochondrial DNA has no effect on its stability
- 2020
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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. ; 117:25, s. 14306-14313
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotides (rNMPs) incorporated in the nuclear genome are a well-established threat to genome stability and can result in DNA strand breaks when not removed in a timely manner. However, the presence of a certain level of rNMPs is tolerated in mitochondrial DNA (mtDNA) although aberrant mtDNA rNMP content has been identified in disease models. We investigated the effect of incorporated rNMPs on mtDNA stability over the mouse life span and found that the mtDNA rNMP content increased during early life. The rNMP content of mtDNA varied greatly across different tissues and was defined by the rNTP/dNTP ratio of the tissue. Accordingly, mtDNA rNMPs were nearly absent in SAMHD1(-/-) mice that have increased dNTP pools. The near absence of rNMPs did not, however, appreciably affect mtDNA copy number or the levels of mtDNA molecules with deletions or strand breaks in aged animals near the end of their life span. The physiological rNMP load therefore does not contribute to the progressive loss of mtDNA quality that occurs as mice age.
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| 20. |
- Wanrooij, Paulina H., et al.
(författare)
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NME6: ribonucleotide salvage sustains mitochondrial transcription
- 2023
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Ingår i: EMBO Journal. - : John Wiley & Sons. - 0261-4189 .- 1460-2075. ; 42:18
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Tidskriftsartikel (refereegranskat)abstract
- The building blocks for RNA and DNA are made in the cytosol, meaning mitochondria depend on the import and salvage of ribonucleoside triphosphates (rNTPs) and deoxyribonucleoside triphosphates (dNTPs) for the synthesis of their own genetic material. While extensive research has focused on mitochondrial dNTP homeostasis due to its defects being associated with various mitochondrial DNA (mtDNA) depletion and deletion syndromes, the investigation of mitochondrial rNTP homeostasis has received relatively little attention. In this issue of the EMBO Journal, Grotehans et al provide compelling evidence of a major role for NME6, a mitochondrial nucleoside diphosphate kinase, in the conversion of pyrimidine ribonucleoside diphosphates into the corresponding triphosphates. These data also suggest a significant physiological role for NME6, as its absence results in the depletion of mitochondrial transcripts and destabilization of the electron transport chain (Grotehans et al, 2023).
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| 21. |
- Wanrooij, Paulina H., et al.
(författare)
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Probing the Mec1ATR Checkpoint Activation Mechanism with Small Peptides
- 2016
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 291:1, s. 393-401
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Tidskriftsartikel (refereegranskat)abstract
- Yeast Mec1, the ortholog of human ATR, is the apical protein kinase that initiates the cell cycle checkpoint in response to DNA damage and replication stress. The basal activity of Mec1 kinase is activated by cell cycle phase-specific activators. Three distinct activators stimulate Mec1 kinase using an intrinsically disordered domain of the protein. These are the Ddc1 subunit of the 9-1-1 checkpoint clamp (ortholog of human and Schizosaccharomyces pombe Rad9), the replication initiator Dpb11 (ortholog of human TopBP1 and S. pombe Cut5), and the multifunctional nuclease/helicase Dna2. Here, we use small peptides to determine the requirements for Mec1 activation. For Ddc1, we identify two essential aromatic amino acids in a hydrophobic environment that when fused together are proficient activators. Using this increased insight, we have been able to identify homologous motifs in S. pombe Rad9 that can activate Mec1. Furthermore, we show that a 9-amino acid Dna2-based peptide is sufficient for Mec1 activation. Studies with mutant activators suggest that binding of an activator to Mec1 is a two-step process, the first step involving the obligatory binding of essential aromatic amino acids to Mec1, followed by an enhancement in binding energy through interactions with neighboring sequences.
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| 22. |
- Wanrooij, Paulina H., et al.
(författare)
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Ribonucleotides in mitochondrial DNA
- 2019
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Ingår i: FEBS Letters. - : John Wiley & Sons. - 0014-5793 .- 1873-3468. ; 593:13, s. 1554-1565
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Forskningsöversikt (refereegranskat)abstract
- The incorporation of ribonucleotides (rNMPs) into DNA during genome replication has gained substantial attention in recent years and has been shown to be a significant source of genomic instability. Studies in yeast and mammals have shown that the two genomes, the nuclear DNA (nDNA) and the mitochondrial DNA (mtDNA), differ with regard to their rNMP content. This is largely due to differences in rNMP repair - whereas rNMPs are efficiently removed from the nuclear genome, mitochondria lack robust mechanisms for removal of single rNMPs incorporated during DNA replication. In this minireview, we describe the processes that determine the frequency of rNMPs in the mitochondrial genome and summarise recent findings regarding the effect of incorporated rNMPs on mtDNA stability and function.
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| 23. |
- Wanrooij, Paulina Hannele
(författare)
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The interface of mitochondrial DNA transcription and replication
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mitochondria are a dynamic network of subcellular organelles that produce the majority of cellular ATP through the process of oxidative phosphorylation (OXPHOS). The components of the respiratory chain are encoded by two separate genomes, nuclear DNA and mitochondrial DNA (mtDNA), and the proper maintenance of both of these genomic entities is therefore crucial for cellular ATP levels and the survival of the cell. Dysfunction of the respiratory chain leads to cellular energy deficiency and mitochondrial disease, which can manifest in a variety of ways but primarily affects tissues of higher energy demand. Although mtDNA replication and transcription are of vital importance for the cell, the molecular mechanisms behind these processes are not fully understood. In mammalian cells, mtDNA replication initiates from two major sites, the origins of heavy and light strand replication (OriH and OriL, respectively). Activation of both origins requires a short RNA primer that is generated by the mitochondrial transcription machinery. In this way, mtDNA replication and transcription are intricately linked. At OriH, primer 3′ end formation has been suggested to rely on nucleolytic processing of full-length transcripts, but only trace amounts of the nuclease implied in this process are found in mitochondria, making this an unlikely model. In this thesis, we demonstrate that the formation of the primer 3′ end is a sequence-dependent event that is directed by the Conserved Sequence Block II (CSBII) sequence element in mtDNA. During transcription of CSBII, the nascent RNA adopts a G-quadruplex structure that causes premature termination of transcription in vitro. After transcription termination, the primer RNA remains stably associated with the DNA in a persistent RNA-DNA hybrid called an R-loop. We find that this interaction is mediated by hybrid Gquadruplex structures that form between the RNA primer and the DNA non-template strand. When G-quadruplex formation in either the RNA transcript or in the DNA is prevented, the stable association of the primer RNA is lost. The mitochondrial RNA polymerase (POLRMT) is also involved in generating the primer at the origin of light strand replication (OriL). In order to define the essential sequence requirements of mammalian mitochondrial OriL, we employ an in vivo saturation mutagenesis approach combined with biochemical analysis. Our results support an essential role of OriL in the mouse, consistent with the strand-displacement model of mtDNA replication. Furthermore, bioinformatic analysis demonstrates conservation of the OriL structure in vertebrates. POLRMT requires two accessory factors for transcription initiation at mitochondrial promoters. However, the necessity of the mitochondrial transcription factor A (TFAM) in this process has been questioned. We use our defined mitochondrial in vitro transcription system to confirm the important role TFAM in transcription initiation. The requirement for TFAM can be circumvented by conditions that promote DNA breathing, such as low salt concentrations or the use of negatively supercoiled template. We demonstrate that TFAM has the capacity to generate negative supercoils, which we speculate may contribute to melting of the promoter.
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