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

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

The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method

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

Mitochondrial membrane potential acts as a retrograde signal to regulate cell cycle progression

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

Inosine Triphosphate Pyrophosphatase Dephosphorylates Ribavirin Triphosphate and Reduced Enzymatic Activity Potentiates Mutagenesis in Hepatitis C Virus

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

Elimination of rNMPs from mitochondrial DNA has no effect on its stability

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