Oxidative DNA damage stalls the human mitochondrial replisome

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 γ 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

Ribonucleotides in mitochondrial DNA

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.Special Issue: Krakow Special Issue</p

NME6: ribonucleotide salvage sustains mitochondrial transcription

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)

DNA Damage Tolerance by Eukaryotic DNA Polymerase and Primase PrimPol

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

Mitochondrial DNA Instability in Mammalian Cells

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

The effect of free rNTPs on the processivity of WT and exo^- Pol γ.

<p>(<b>A</b>) Schematic diagram of the processivity assay carried out with WT and exo^- Pol γ on a primed, circular 3 kb template in the presence and absence of rNTPs. (<b>B</b>) Analysis of the processivity of WT and exo^- Pol γ as depicted in Fig 4A. Reactions were stopped after 10, 30, 60, and 90 min and the DNA products were analysed by agarose gel electrophoresis. All reactions were carried out in the presence of 750 nM mtSSB and 10 μM dNTPs. Where indicated, rNTPs were added. Fig 4B shows a representative figure of five (WT) and three (exo^-) independent experiments. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007315#pgen.1007315.s004" target="_blank">S4 Fig</a>. (<b>C</b>) A plot of the signal intensity of the full-length product at each time point in Fig 4B. (<b>D</b>) Processivity was tested during an increasing amount of dNTPs (0, 2, 5, 10, 25 or 50 μM) with or without rNTPs at fixed concentration. Reactions were stopped after 60 min and analysed on TBE agarose gel. (<b>E</b>) To stimulate the conditions of imbalanced dNTP pools, each dNTP was limited to 1 μM (indicated dA, dG, dC or dT) and compared with “normal” dNTP conditions (indicated as N). Reactions run for 60 min, with or without rNTPs present and analysed on TBE agarose gel.</p

The influence of exonuclease activity on rNMP incorporation by Pol γ.

<p>(<b>A</b>) Schematic view of reaction set up in Fig 3B. (<b>B</b>) Comparison of rNMP incorporation frequencies of wild type (WT) and exonuclease deficient (exo^-) Pol γ. <i>In vitro</i> replication of a primed 7.3 kb ssDNA template was performed with 10 μM dNTPs in the presence (+) or absence (-) of rNTPs. Reaction products were followed by addition of [α-³²P]-dCTP. Samples were alkaline-treated (“+ NaOH” lanes 5–8) or untreated control (“-NaOH” lanes 1–4) for 2 h at 55°C and run on a denaturing alkaline gel. Fig 3B is a representative picture of three independent experiments. (<b>C</b>) Distribution plot of the percentage of total signal intensity from NaOH-treated samples in Fig 3B. The curves for WT (black and grey) and exo^- (brown and orange) Pol γ overlap, which indicates a similar incorporation frequency. (<b>D</b>) Southern blot analysis against the 16S rDNA region of mtDNA isolated from the liver of WT <i>PolgA</i> (n = 2) and exonuclease-deficient <i>PolgA</i>^<i>D257A</i> (n = 3; “exo^-”) mice. SacI-linearized mtDNA was treated with alkaline hydrolysis (“A”) and run on an alkaline gel alongside untreated control samples (“C”). (<b>E</b>) Distribution plot of the DNA fragments in control and alkaline-treated samples from Fig 3D. The comparable distribution of DNA fragment size after alkaline-treatment is consistent with a comparable rNMP incorporation frequency in liver mtDNA of WT <i>PolgA</i> and <i>PolgA</i>^<i>D257A</i> mice.</p

rNMP incorporation frequency of Pol γ and the mtDNA replisome on long DNA templates.

<p>(<b>A</b>) Schematic diagram of the 7.3 kb M13 ssDNA substrate used to compare the incorporation of rNMPs by Pol γ and yeast Pol δ in the primer extension assay in Fig 2B. The newly synthesized DNA was labelled by addition of [α-³²P]-dCTP to the reaction. (<b>B</b>) Analysis of the rNMP incorporation frequency of Pol γ and yeast Pol δ at “normal” (“N”, S phase) concentrations, “low” (“L”, concentration during the rest of the cell cycle and in non-dividing cells), or <i>S</i>. <i>cerevisiae</i> (“Sc”) dNTP concentrations, in the absence or presence of rNTPs. In all reactions, the DNA template was coated with the relevant single-stranded DNA-binding proteins (lanes 1–6 with RPA; lanes 7–14 with mtSSB) to avoid stalling of DNA synthesis due to formation of DNA secondary structures. Untreated (“-NaOH”) or alkaline (“+NaOH”) treated reaction products were analysed on an agarose gel under denaturing conditions. To estimate rNMP incorporation frequencies from the presented gel, the median length of alkali stable products was determined and used to calculate the frequencies as described in Materials and Methods. Numbers on the left-hand side of the gel indicate positions of DNA marker bands and the full-length starting product (7.3) in kb. The gel is a representative picture of four independent experiments. (<b>C</b>) Schematic diagram of the primed mini-circle substrate with a 40 nt 5′ overhang used in Fig 2D. (<b>D</b>) Analysis of the rNMP incorporation frequency by the mitochondrial replisome consisting of mtSSB, Twinkle and Pol γ (AB₂) on a primed mini-circle substrate with a 5′ overhang for Twinkle loading. Reactions were carried out at normal (“N), low (“L”), and “<i>S</i>. <i>cerevisiae</i>” (“Sc”) dNTP concentrations in the presence or absence of rNTPs. Untreated (“-NaOH”) and alkaline (“+NaOH”) treated reaction products were analysed on a denaturing (alkaline) agarose gel. The gel is a representative picture of two independent experiments.</p