Linear DNA-driven recombination in mammalian mitochondria.

Mitochondrial DNA (mtDNA) recombination in animals has remained enigmatic due to its uniparental inheritance and subsequent homoplasmic state, which excludes the biological need for genetic recombination, as well as limits tools to study it. However, molecular recombination is an important genome maintenance mechanism for all organisms, most notably being required for double-strand break repair. To demonstrate the existence of mtDNA recombination, we took advantage of a cell model with two different types of mitochondrial genomes and impaired its ability to degrade broken mtDNA. The resulting excess of linear DNA fragments caused increased formation of cruciform mtDNA, appearance of heterodimeric mtDNA complexes and recombinant mtDNA genomes, detectable by Southern blot and by long range PacBio® HiFi sequencing approach. Besides utilizing different electrophoretic methods, we also directly observed molecular complexes between different mtDNA haplotypes and recombination intermediates using transmission electron microscopy. We propose that the known copy-choice recombination by mitochondrial replisome could be sufficient for the needs of the small genome, thus removing the requirement for a specialized mitochondrial recombinase. The error-proneness of this system is likely to contribute to the formation of pathological mtDNA rearrangements.

Uncharacterized protein C17orf80 - a novel interactor of human mitochondrial nucleoids.

Molecular functions of many human proteins remain unstudied, despite the demonstrated association with diseases or pivotal molecular structures, such as mitochondrial DNA (mtDNA). This small genome is crucial for the proper functioning of mitochondria, the energy-converting organelles. In mammals, mtDNA is arranged into macromolecular complexes called nucleoids that serve as functional stations for its maintenance and expression. Here, we aimed to explore an uncharacterized protein C17orf80, which was previously detected close to the nucleoid components by proximity labelling mass spectrometry. To investigate the subcellular localization and function of C17orf80, we took advantage of immunofluorescence microscopy, interaction proteomics and several biochemical assays. We demonstrate that C17orf80 is a mitochondrial membrane-associated protein that interacts with nucleoids even when mtDNA replication is inhibited. In addition, we show that C17orf80 is not essential for mtDNA maintenance and mitochondrial gene expression in cultured human cells. These results provide a basis for uncovering the molecular function of C17orf80 and the nature of its association with nucleoids, possibly leading to new insights about mtDNA and its expression.

Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells.

Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.

Analysis of Mitochondrial DNA Replication by Two-Dimensional Agarose Gel Electrophoresis.

Two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE) has been employed for nearly two decades in the analysis of replication and maintenance processes of animal mitochondrial DNA, but the method's potential has not been fully exploited. Here, we describe the various steps involved in this technique, from DNA isolation, to two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE), Southern hybridization and interpretation. We also provide examples of the applicability of 2D-AGE to investigate the different features of mtDNA maintenance and regulation.

Molecular phenotyping uncovers differences in basic housekeeping functions among closely related species of hares (Lepus spp., Lagomorpha: Leporidae).

Speciation is a fundamental evolutionary process, which results in genetic differentiation of populations and manifests as discrete morphological, physiological and behavioural differences. Each species has travelled its own evolutionary trajectory, influenced by random drift and driven by various types of natural selection, making the association of genetic differences between the species with the phenotypic differences extremely complex to dissect. In the present study, we have used an in vitro model to analyse in depth the genetic and gene regulation differences between fibroblasts of two closely related mammals, the arctic/subarctic mountain hare (Lepus timidus Linnaeus) and the temperate steppe-climate adapted brown hare (Lepus europaeus Pallas). We discovered the existence of a species-specific expression pattern of 1623 genes, manifesting in differences in cell growth, cell cycle control, respiration, and metabolism. Interspecific differences in the housekeeping functions of fibroblast cells suggest that speciation acts on fundamental cellular processes, even in these two interfertile species. Our results help to understand the molecular constituents of a species difference on a cellular level, which could contribute to the maintenance of the species boundary.

Draft Genome Sequence of Bradyrhizobium denitrificans K2, Cultivated from an Air Ventilation Environment.

Bradyrhizobium denitrificans K2, isolated from an air circulation environment, has potential genes participating in inorganic nitrogen and carbon cycling. The draft genome comprises 8.31 Mb, with 7,982 coding sequences and 64.81% average G+C content. Genes related to carbon and inorganic nitrogen cycling were observed in the draft genome.

Top3α is the replicative topoisomerase in mitochondrial DNA replication.

Mitochondrial DNA has been investigated for nearly fifty years, but many aspects of the maintenance of this essential small genome remain unknown. Like any genome, mammalian mitochondrial DNA requires the function of topoisomerases to counter and regulate the topological tension arising during replication, transcription, segregation, and repair. However, the functions of the different mitochondrial topoisomerases are poorly understood. Here, we investigate the role of Topoisomerase 3α (Top3α) in mtDNA replication and transcription, providing evidence that this enzyme, previously reported to act in mtDNA segregation, also participates in mtDNA replication fork progression. Top3α knockdown caused replication fork stalling, increased mtDNA catenation and decreased mtDNA levels. Overexpression in contrast induced abundant double-strand breaks around the replication origin OH and abortion of early replication, while at the same time improving the resolution of mtDNA replication termination intermediates. Both Top3α knockdown and overexpression affected mitochondrial RNA transcription, leading to a decrease in steady-state levels of mitochondrial transcripts. Together, our results indicate that the mitochondrial isoform of Top3α is not only involved in mtDNA segregation, as reported previously, but also supports the progression of the replication fork. Mitochondrial Top3α is also influencing the progression of transcription, with its absence affecting downstream transcript levels.

Heterozygous p.Y955C mutation in DNA polymerase γ leads to alterations in bioenergetics, complex I subunit expression, and mtDNA replication.

In human cells, ATP is generated using oxidative phosphorylation machinery, which is inoperable without proteins encoded by mitochondrial DNA (mtDNA). The DNA polymerase gamma (Polγ) repairs and replicates the multicopy mtDNA genome in concert with additional factors. The Polγ catalytic subunit is encoded by the POLG gene, and mutations in this gene cause mtDNA genome instability and disease. Barriers to studying the molecular effects of disease mutations include scarcity of patient samples and a lack of available mutant models; therefore, we developed a human SJCRH30 myoblast cell line model with the most common autosomal dominant POLG mutation, c.2864A>G/p.Y955C, as individuals with this mutation can present with progressive skeletal muscle weakness. Using on-target sequencing, we detected a 50% conversion frequency of the mutation, confirming heterozygous Y955C substitution. We found mutated cells grew slowly in a glucose-containing medium and had reduced mitochondrial bioenergetics compared with the parental cell line. Furthermore, growing Y955C cells in a galactose-containing medium to obligate mitochondrial function enhanced these bioenergetic deficits. Also, we show complex I NDUFB8 and ND3 protein levels were decreased in the mutant cell line, and the maintenance of mtDNA was severely impaired (i.e., lower copy number, fewer nucleoids, and an accumulation of Y955C-specific replication intermediates). Finally, we show the mutant cells have increased sensitivity to the mitochondrial toxicant 2'-3'-dideoxycytidine. We expect this POLG Y955C cell line to be a robust system to identify new mitochondrial toxicants and therapeutics to treat mitochondrial dysfunction.

Adaptive and Pathological Outcomes of Radiation Stress-Induced Redox Signaling.

Significance: Ionizing radiation can damage cells either directly or through oxidative damage caused by ionization. Although radiation exposure from natural sources is very limited, ionizing radiation in nuclear disaster zones and long spaceflights causes inconspicuous, yet measurable physiological effects in men and animals, whose significance remains poorly known. Understanding the physiological impacts of ionizing radiation has a wide importance due to the increased use of medical imaging and radiotherapy. Recent Advances: Radiation exposure has been traditionally investigated from the perspective of DNA damage and its consequences. However, recent studies from Chernobyl as well as spaceflights have provided interesting insights into oxidative stress-induced metabolic alterations and disturbances in the circadian regulation. Critical Issues: In this review, we discuss the physiological consequences of radiation exposure in the light of oxidative stress signaling. Radiation exposure likely triggers many converging or interconnecting signaling pathways, some of which mimic mitochondrial dysfunction and might explain the observed metabolic changes. Future Directions: Better understanding of the different radiation-induced signaling pathways might help to devise strategies for mitigation of the long-term effects of radiation exposure. The utility of fibroblast growth factor 21 (FGF21) as a radiation exposure biomarker and the use of radiation hormesis as a method to protect astronauts on a prolonged spaceflight, such as a mission to Mars, should be investigated. Antioxid. Redox Signal. 37, 336-348.

MRE11-dependent instability in mitochondrial DNA fork protection activates a cGAS immune signaling pathway.

Mitochondrial DNA (mtDNA) instability activates cGAS-dependent innate immune signaling by unknown mechanisms. Here, we find that Fanconi anemia suppressor genes are acting in the mitochondria to protect mtDNA replication forks from instability. Specifically, Fanconi anemia patient cells show a loss of nascent mtDNA through MRE11 nuclease degradation. In contrast to DNA replication fork stability, which requires pathway activation by FANCD2-FANCI monoubiquitination and upstream FANC core complex genes, mitochondrial replication fork protection does not, revealing a mechanistic and genetic separation between mitochondrial and nuclear genome stability pathways. The degraded mtDNA causes hyperactivation of cGAS-dependent immune signaling resembling the unphosphorylated ISG3 response. Chemical inhibition of MRE11 suppresses this innate immune signaling, identifying MRE11 as a nuclease responsible for activating the mtDNA-dependent cGAS/STING response. Collective results establish a previously unknown molecular pathway for mtDNA replication stability and reveal a molecular handle to control mtDNA-dependent cGAS activation by inhibiting MRE11 nuclease.

TFAM knockdown-triggered mtDNA-nucleoid aggregation and a decrease in mtDNA copy number induce the reorganization of nucleoid populations and mitochondria-associated ER-membrane contacts.

The correct organization of mitochondrial DNA (mtDNA) in nucleoids and the contacts of mitochondria with the ER play an important role in maintaining the mitochondrial genome distribution within the cell. Mitochondria-associated ER membranes (MAMs) consist of interacting proteins and lipids located in the outer mitochondrial membrane and ER membrane, forming a platform for the mitochondrial inner membrane-associated genome replication factory as well as connecting the nucleoids with the mitochondrial division machinery. We show here that knockdown of a core component of mitochondrial nucleoids, TFAM, causes changes in the mitochondrial nucleoid populations, which subsequently impact ER-mitochondria membrane contacts. Knockdown of TFAM causes a significant decrease in the copy number of mtDNA as well as aggregation of mtDNA nucleoids. At the same time, it causes significant upregulation of the replicative TWNK helicase in the membrane-associated nucleoid fraction. This is accompanied by a transient elevation of MAM proteins, indicating a rearrangement of the linkage between ER and mitochondria triggered by changes in mitochondrial nucleoids. Reciprocal knockdown of the mitochondrial replicative helicase TWNK causes a decrease in mtDNA copy number and modifies mtDNA membrane association, however, it does not cause nucleoid aggregation and considerable alterations of MAM proteins in the membrane-associated fraction. Our explanation is that the aggregation of mitochondrial nucleoids resulting from TFAM knockdown triggers a compensatory mechanism involving the reorganization of both mitochondrial nucleoids and MAM. These results could provide an important insight into pathological conditions associated with impaired nucleoid organization or defects of mtDNA distribution.

The Type and Source of Reactive Oxygen Species Influences the Outcome of Oxidative Stress in Cultured Cells.

Oxidative stress can be modeled using various different experimental approaches, such as exposing the cells or organisms to oxidative chemicals. However, the actual effects of these chemicals, outside of the immediate measured effect, have attracted relatively little attention. We show here that three commonly used oxidants, menadione, potassium bromate, and hydrogen peroxide, while known to function differently, also elicit different types of responses in HEK293T cells. Menadione and bromate exposure mainly trigger an integrated stress response, whereas hydrogen peroxide affects cellular processes more diversely. Interestingly, acute oxidative stress does not universally cause notable induction of DNA repair or antioxidant defense mechanisms. We also provide evidence that cells with previous experience of oxidative stress show adaptive changes in their responses when the stress is renewed. Our results urge caution when comparing studies where different sources of oxidative stress have been used or when generalizing the findings of these studies to other oxidant types or tissues.

Author Correction: Defects in mtDNA replication challenge nuclear genome stability through nucleotide depletion and provide a unifying mechanism for mouse progerias.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Replication fork rescue in mammalian mitochondria.

Replication stalling has been associated with the formation of pathological mitochondrial DNA (mtDNA) rearrangements. Yet, almost nothing is known about the fate of stalled replication intermediates in mitochondria. We show here that replication stalling in mitochondria leads to replication fork regression and mtDNA double-strand breaks. The resulting mtDNA fragments are normally degraded by a mechanism involving the mitochondrial exonuclease MGME1, and the loss of this enzyme results in accumulation of linear and recombining mtDNA species. Additionally, replication stress promotes the initiation of alternative replication origins as an apparent means of rescue by fork convergence. Besides demonstrating an interplay between two major mechanisms rescuing stalled replication forks - mtDNA degradation and homology-dependent repair - our data provide evidence that mitochondria employ similar mechanisms to cope with replication stress as known from other genetic systems.

Twist and Turn-Topoisomerase Functions in Mitochondrial DNA Maintenance.

Like any genome, mitochondrial DNA (mtDNA) also requires the action of topoisomerases to resolve topological problems in its maintenance, but for a long time, little was known about mitochondrial topoisomerases. The last years have brought a closer insight into the function of these fascinating enzymes in mtDNA topology regulation, replication, transcription, and segregation. Here, we summarize the current knowledge about mitochondrial topoisomerases, paying special attention to mammalian mitochondrial genome maintenance. We also discuss the open gaps in the existing knowledge of mtDNA topology control and the potential involvement of mitochondrial topoisomerases in human pathologies. While Top1mt, the only exclusively mitochondrial topoisomerase in mammals, has been studied intensively for nearly a decade, only recent studies have shed some light onto the mitochondrial function of Top2ß and Top3α, enzymes that are shared between nucleus and mitochondria. Top3α mediates the segregation of freshly replicated mtDNA molecules, and its dysfunction leads to mtDNA aggregation and copy number depletion in patients. Top2ß, in contrast, regulates mitochondrial DNA replication and transcription through the alteration of mtDNA topology, a fact that should be acknowledged due to the frequent use of Topoisomerase 2 inhibitors in medical therapy.

Tissue specific differences in mitochondrial DNA maintenance and expression.

The different cell types of multicellular organisms have specialized physiological requirements, affecting also their mitochondrial energy production and metabolism. The genome of mitochondria is essential for mitochondrial oxidative phosphorylation (OXHPOS) and thus plays a central role in many human mitochondrial pathologies. Disorders affecting mitochondrial DNA (mtDNA) maintenance are typically resulting in a tissue-specific pattern of mtDNA deletions and rearrangements. Despite this role in disease as well as a biomarker of mitochondrial biogenesis, the tissue-specific parameters of mitochondrial DNA maintenance have been virtually unexplored. In the presented study, we investigated mtDNA replication, topology, gene expression and damage in six different tissues of adult mice and sought to correlate these with the levels of known protein factors involved in mtDNA replication and transcription. Our results show that while liver and kidney cells replicate their mtDNA using the asynchronous mechanism known from cultured cells, tissues with high OXPHOS activity, such as heart, brain, skeletal muscle and brown fat, employ a strand-coupled replication mode, combined with increased levels of recombination. The strand-coupled replication mode correlated also with mtDNA damage levels, indicating that the replication mechanism represents a tissue-specific strategy to deal with intrinsic oxidative stress. While the preferred replication mode did not correlate with mtDNA transcription or the levels of most known mtDNA maintenance proteins, mtSSB was most abundant in tissues using strand-asynchronous mechanism. Although mitochondrial transcripts were most abundant in tissues with high metabolic rate, the mtDNA copy number per tissue mass was remarkably similar in all tissues. We propose that the tissue-specific features of mtDNA maintenance are primarily driven by the intrinsic reactive oxygen species exposure, mediated by DNA repair factors, whose identity remains to be elucidated.

Defects in mtDNA replication challenge nuclear genome stability through nucleotide depletion and provide a unifying mechanism for mouse progerias.

Mitochondrial DNA (mtDNA) mutagenesis and nuclear DNA repair defects are considered cellular mechanisms of ageing. mtDNA mutator mice with increased mtDNA mutagenesis show signs of premature ageing. However, why patients with mitochondrial diseases, or mice with other forms of mitochondrial dysfunction, do not age prematurely remains unknown. Here, we show that cells from mutator mice display challenged nuclear genome maintenance similar to that observed in progeric cells with defects in nuclear DNA repair. Cells from mutator mice show slow nuclear DNA replication fork progression, cell cycle stalling and chronic DNA replication stress, leading to double-strand DNA breaks in proliferating progenitor or stem cells. The underlying mechanism involves increased mtDNA replication frequency, sequestering of nucleotides to mitochondria, depletion of total cellular nucleotide pools, decreased deoxynucleoside 5'-triphosphate (dNTP) availability for nuclear genome replication and compromised nuclear genome maintenance. Our data indicate that defects in mtDNA replication can challenge nuclear genome stability. We suggest that defects in nuclear genome maintenance, particularly in the stem cell compartment, represent a unified mechanism for mouse progerias. Therefore, through their destabilizing effects on the nuclear genome, mtDNA mutations are indirect contributors to organismal ageing, suggesting that the direct role of mtDNA mutations in driving ageing-like symptoms might need to be revisited.

Ciprofloxacin impairs mitochondrial DNA replication initiation through inhibition of Topoisomerase 2.

Maintenance of topological homeostasis is vital for gene expression and genome replication in all organisms. Similar to other circular genomes, also mitochondrial DNA (mtDNA) is known to exist in various different topological forms, although their functional significance remains unknown. We report here that both known type II topoisomerases Top2α and Top2ß are present in mammalian mitochondria, with especially Top2ß regulating the supercoiling state of mtDNA. Loss of Top2ß or its inhibition by ciprofloxacin results in accumulation of positively supercoiled mtDNA, followed by cessation of mitochondrial transcription and replication initiation, causing depletion of mtDNA copy number. These mitochondrial effects block both cell proliferation and differentiation, possibly explaining some of the side effects associated with fluoroquinolone antibiotics. Our results show for the first time the importance of topology for maintenance of mtDNA homeostasis and provide novel insight into the mitochondrial effects of fluoroquinolones.

Known Unknowns of Mammalian Mitochondrial DNA Maintenance.

Mammalian mitochondrial DNA (mtDNA) replication and repair have been studied intensively for the last 50 years. Although recently advances in elucidating the molecular mechanisms of mtDNA maintenance and the proteins involved in these have been made, there are disturbing gaps between the existing theoretical models and experimental observations. Conflicting data and hypotheses exist about the role of RNA and ribonucleotides in mtDNA replication, but also about the priming of replication and the formation of pathological rearrangements. In the presented review, we have attempted to match these loose ends and draft consensus where it can be found, while identifying outstanding issues for future research.