bims-midmar Biomed News
on Mitochondrial DNA maintenance and replication
Issue of 2022–04–03
fiveteen papers selected by
Flavia Söllner, Ludwig-Maximilians University



  1. Biochim Biophys Acta Bioenerg. 2022 Mar 24. pii: S0005-2728(22)00023-8. [Epub ahead of print]1863(5): 148554
      Mitochondria is a unique cellular organelle involved in multiple cellular processes and is critical for maintaining cellular homeostasis. This semi-autonomous organelle contains its circular genome - mtDNA (mitochondrial DNA), that undergoes continuous cycles of replication and repair to maintain the mitochondrial genome integrity. The majority of the mitochondrial genes, including mitochondrial replisome and repair genes, are nuclear-encoded. Although the repair machinery of mitochondria is quite efficient, the mitochondrial genome is highly susceptible to oxidative damage and other types of exogenous and endogenous agent-induced DNA damage, due to the absence of protective histones and their proximity to the main ROS production sites. Mutations in replication and repair genes of mitochondria can result in mtDNA depletion and deletions subsequently leading to mitochondrial genome instability. The combined action of mutations and deletions can result in compromised mitochondrial genome maintenance and lead to various mitochondrial disorders. Here, we review the mechanism of mitochondrial DNA replication and repair process, key proteins involved, and their altered function in mitochondrial disorders. The focus of this review will be on the key genes of mitochondrial DNA replication and repair machinery and the clinical phenotypes associated with mutations in these genes.
    Keywords:  Base-excision repair; CPEO; LIG3; POLG; mtDNA replication
    DOI:  https://doi.org/10.1016/j.bbabio.2022.148554
  2. J Vis Exp. 2022 Mar 09.
      Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human mitochondrial disorders. From a genetic point of view, the OXPHOS represents a unique example because it results from the complementation of two distinct genetic systems: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, OXPHOS defects can be due to mutations affecting nuclear and mitochondrial encoded genes. The groundbreaking work by King and Attardi, published in 1989, showed that human cell lines depleted of mtDNA (named rho0) could be repopulated by exogenous mitochondria to obtain the so-called "transmitochondrial cybrids." Thanks to these cybrids containing mitochondria derived from patients with mitochondrial disorders (MDs) and nuclei from rho0 cells, it is possible to verify whether a defect is mtDNA- or nDNA-related. These cybrids are also a powerful tool to validate the pathogenicity of a mutation and study its impact at a biochemical level. This paper presents a detailed protocol describing cybrid generation, selection, and characterization.
    DOI:  https://doi.org/10.3791/63452
  3. Biol Chem. 2022 Mar 31.
      Mitochondria are central hubs for cellular metabolism, coordinating a variety of metabolic reactions crucial for human health. Mitochondria provide most of the cellular energy via their oxidative phosphorylation (OXPHOS) system, which requires the coordinated expression of genes encoded by both the nuclear (nDNA) and mitochondrial genomes (mtDNA). Transcription of mtDNA is not only essential for the biogenesis of the OXPHOS system, but also generates RNA primers necessary to initiate mtDNA replication. Like the prokaryotic system, mitochondria have no membrane-based compartmentalization to separate the different steps of mtDNA maintenance and expression and depend entirely on nDNA-encoded factors imported into the organelle. Our understanding of mitochondrial transcription in mammalian cells has largely progressed, but the mechanisms regulating mtDNA gene expression are still poorly understood despite their profound importance for human disease. Here, we review mechanisms of mitochondrial gene expression with a focus on the recent findings in the field of mammalian mtDNA transcription and disease phenotypes caused by defects in proteins involved in this process.
    Keywords:   inhibitor of mitochondrial transcription; PPR proteins; mitochondria; mitochondrial disease; mitochondrial gene expression; mitochondrial transcription
    DOI:  https://doi.org/10.1515/hsz-2021-0416
  4. Front Genet. 2022 ;13 692257
      Mitochondrial DNA (mtDNA) mutations contribute to human disease across a range of severity, from rare, highly penetrant mutations causal for monogenic disorders to mutations with milder contributions to phenotypes. mtDNA variation can exist in all copies of mtDNA or in a percentage of mtDNA copies and can be detected with levels as low as 1%. The large number of copies of mtDNA and the possibility of multiple alternative alleles at the same DNA nucleotide position make the task of identifying allelic variation in mtDNA very challenging. In recent years, specialized variant calling algorithms have been developed that are tailored to identify mtDNA variation from whole-genome sequencing (WGS) data. However, very few studies have systematically evaluated and compared these methods for the detection of both homoplasmy and heteroplasmy. A publicly available synthetic gold standard dataset was used to assess four mtDNA variant callers (Mutserve, mitoCaller, MitoSeek, and MToolBox), and the commonly used Genome Analysis Toolkit "best practices" pipeline, which is included in most current WGS pipelines. We also used WGS data from 126 trios and calculated the percentage of maternally inherited variants as a metric of calling accuracy, especially for homoplasmic variants. We additionally compared multiple pathogenicity prediction resources for mtDNA variants. Although the accuracy of homoplasmic variant detection was high for the majority of the callers with high concordance across callers, we found a very low concordance rate between mtDNA variant callers for heteroplasmic variants ranging from 2.8% to 3.6%, for heteroplasmy thresholds of 5% and 1%. Overall, Mutserve showed the best performance using the synthetic benchmark dataset. The analysis of mtDNA pathogenicity resources also showed low concordance in prediction results. We have shown that while homoplasmic variant calling is consistent between callers, there remains a significant discrepancy in heteroplasmic variant calling. We found that resources like population frequency databases and pathogenicity predictors are now available for variant annotation but still need refinement and improvement. With its peculiarities, the mitochondria require special considerations, and we advocate that caution needs to be taken when analyzing mtDNA data from WGS data.
    Keywords:  benchmarking; heteroplasmic; homoplasmic; mitochondrial DNA; variant‐caller; whole-genome sequencing
    DOI:  https://doi.org/10.3389/fgene.2022.692257
  5. Front Neurol. 2022 ;13 846110
      Mutations in nuclear-encoded genes that are involved in mitochondrial DNA replication and maintenance (e.g., POLG) have been associated with chronic progressive external ophthalmoplegia (CPEO) phenotype. These nuclear genome mutations may lead to multiple mitochondrial DNA deletions or mitochondrial DNA depletion. On the other hand, primary genetic defects of mitochondrial DNA (such as single large-scale deletion or point mutations) have also been associated with the CPEO phenotype. Chronic progressive external ophthalmoplegia (CPEO) may be a manifestation of specific syndromes that, when clinically recognized, prompt clinicians to investigate specific genetic defects. Thus, CPEO, as part of Kearns Sayre syndrome, suggests the presence of a large-scale deletion of mitochondrial DNA. However, in pure CPEO or CPEO plus phenotypes, it is more difficult to know whether causative genetic defects affect the nuclear or mitochondrial DNA. Here, we present a patient with a long-standing history of CPEO plus phenotype, in whom the sequencing of mitochondrial DNA from skeletal muscle was normal, and no other genetic defect was suspected at first. At the time of our evaluation, the presence of polyneuropathy and neuropathic pain prompted us to investigate nuclear genetic defects and, specifically, mutations in the POLG gene. Thus, the sequencing of the POLG gene revealed p.Thr251Ile and p.Pro587Leu mutations in one allele, and p.Ala467Thr mutation in another allele. Although one would expect that mutations in POLG lead to multiple mitochondrial DNA deletions or depletion (loss of copies), the absence of mitochondrial DNA abnormalities in tissue may be explained by heteroplasmy, a lack or no significant involvement of biopsied tissue, or a sampling bias. So, the absence of secondary mitochondrial DNA alterations should not discourage clinicians from further investigating mutations in nuclear-encoded genes. Lastly, mitochondrial point mutations and single mitochondrial DNA deletions very rarely cause CPEO associated with polyneuropathy and neuropathic pain, and POLG-related disease should be considered in this scenario, instead.
    Keywords:  CPEO; POLG; mitochondrial disease; neuropathic pain; polyneuropathy
    DOI:  https://doi.org/10.3389/fneur.2022.846110
  6. PLoS Genet. 2022 Apr 01. 18(4): e1010068
      Mitochondria are implicated in the pathogenesis of cardiovascular diseases (CVDs) but the reasons for this are not well understood. Maternally-inherited population variants of mitochondrial DNA (mtDNA) which affect all mtDNA molecules (homoplasmic) are associated with cardiometabolic traits and the risk of developing cardiovascular disease. However, it is not known whether mtDNA mutations only affecting a proportion of mtDNA molecules (heteroplasmic) also play a role. To address this question, we performed a high-depth (~1000-fold) mtDNA sequencing of blood DNA in 1,399 individuals with hypertension (HTN), 1,946 with ischemic heart disease (IHD), 2,146 with ischemic stroke (IS), and 723 healthy controls. We show that the per individual burden of heteroplasmic single nucleotide variants (mtSNVs) increases with age. The age-effect was stronger for low-level heteroplasmies (heteroplasmic fraction, HF, 5-10%), likely reflecting acquired somatic events based on trinucleotide mutational signatures. After correcting for age and other confounders, intermediate heteroplasmies (HF 10-95%) were more common in hypertension, particularly involving non-synonymous variants altering the amino acid sequence of essential respiratory chain proteins. These findings raise the possibility that heteroplasmic mtSNVs in the pathophysiology of hypertension.
    DOI:  https://doi.org/10.1371/journal.pgen.1010068
  7. Biomed Res Int. 2022 ;2022 5344418
      Mitochondrial DNA haplogroup classification is used to study maternal lineage of ancient human populations. The haplogrouping of ancient DNA is not easy because the DNA is usually found in small pieces in limited quantities. We have developed Haplotracker, a straightforward and efficient high-resolution haplogroup classification tool optimized specifically for ancient DNA samples. Haplotracker offers a user-friendly input interface for multiple mitochondrial DNA sequence fragments in a sample. It provides accurate haplogroup classification with full-length mitochondrial genome sequences and provides high-resolution haplogroup predictions for some fragmented control region sequences using a novel algorithm built on Phylotree mtDNA Build 17 (Phylotree) and our haplotype database (n = 118,869). Its performance for accuracy was demonstrated to be high through haplogroup classification using 8,216 Phylotree full-length and control region mitochondrial DNA sequences compared with HaploGrep 2, one of the most accurate current haplogroup classifiers. Haplotracker provides a novel haplogroup tracking solution for fragmented sequences to track subhaplogroups or verify the haplogroups efficiently. Using Haplotracker, we classified mitochondrial haplogroups to the final subhaplogroup level in nine ancient DNA samples extracted from human skeletal remains found in 2,000-year-old elite Xiongnu cemetery in Northeast Mongolia. Haplotracker can be freely accessed at https://haplotracker.cau.ac.kr.
    DOI:  https://doi.org/10.1155/2022/5344418
  8. Front Mol Biosci. 2022 ;9 808036
      Human mitochondrial DNA contains more UV-induced lesions than the nuclear DNA due to lack of mechanism to remove bulky photoproducts. Human DNA polymerase gamma (Pol γ) is the sole DNA replicase in mitochondria, which contains a polymerase (pol) and an exonuclease (exo) active site. Previous studies showed that Pol γ only displays UV lesion bypassing when its exonuclease activity is obliterated. To investigate the reaction environment on Pol γ translesion activity, we tested Pol γ DNA activity in the presence of different metal ions. While Pol γ is unable to replicate through UV lesions on DNA templates in the presence of Mg2+, it exhibits robust translesion DNA synthesis (TLS) on cyclobutane pyrimidine dimer (CPD)-containing template when Mg2+ was mixed with or completely replaced by Mn2+. Under these conditions, the efficiency of Pol γ's TLS opposite CPD is near to that on a non-damaged template and is 800-fold higher than that of exonuclease-deficient Pol γ. Interestingly, Pol γ exhibits higher exonuclease activity in the presence of Mn2+ than with Mg2+, suggesting Mn2+-stimulated Pol γ TLS is not via suppressing its exonuclease activity. We suggest that Mn2+ ion expands Pol γ's pol active site relative to Mg2+ so that a UV lesion can be accommodated and blocks the communication between pol and exo active sites to execute translesion DNA synthesis.
    Keywords:  DNA polymerase gamma; TLS; UV lesion; metal-dependence; mitochondrial DNA
    DOI:  https://doi.org/10.3389/fmolb.2022.808036
  9. Cancer Discov. 2021 Dec 01. 11(12): 2965-2967
       SUMMARY: In this issue of Cancer Discovery, Penter and colleagues describe the results of applying the recently described technology of combined assay for transposase-accessible chromatin using sequencing and mitochondrial DNA sequencing in single cells from serial samples from nine patients with chronic lymphocytic leukemia. The naturally occurring barcodes, provided by mitochondrial DNA mutations, allowed tracking of subclones with distinct chromatin accessibility profiles and copy-number alterations demonstrating distinct patterns of tumor evolution under a range of selection pressures. See related article by Penter et al., p. 3048.
    DOI:  https://doi.org/10.1158/2159-8290.CD-21-1225
  10. Chembiochem. 2022 Mar 28.
      Mitochondrion is the core site of cell signaling, energy metabolism and biosynthesis. Here, taking advantage of activitybased probes, we synthesized two photocontrollable probes ( YGH-1 and YGH-2 ), composed of a mitochondrial localization moiety "triphenylphosphonium", a photo triggered group to achieve spatial and temporal controlled protein capture and an alkyne group to enrich the labeled protein. Proteomic validation was further carried out to facilitate identifications of mitochondrial proteomes in HeLa cells. The results showed that half of identified protein hits (~300) labeled by probes YGH-1 and YGH-2 belong to mitochondria, mostly localizing in mitochondrial matrix and inner mitochondrial membrane. Our research results provide a new tool for spatial and temporal analysis of subcellular proteome.
    Keywords:  mitochondrial proteome; photocontrollable; photocross linker; quinone methide
    DOI:  https://doi.org/10.1002/cbic.202200066
  11. MethodsX. 2022 ;9 101651
      Macrophages are phagocytic cells from the innate immune system that are critical for tissue homeostasis and form the first line of host defense against invading pathogens. The zebrafish larva is an exquisite model to decipher the transcriptional response of macrophages after injury. We used a macrophage reporter line in which an mfap4 promoter drives the expression of a farnesylated mCherry fluorescent protein to label macrophages and we performed tissue dissociation, cell isolation by Fluorescence Activated Cell sorting and RNA preparation. The two bottlenecks are (i) the dissociation of the embryos that often relies on cell suspension steps that alter the activation status of immune cells, and (ii) obtaining high RNA integrity for gene expression analysis from a small number of isolated macrophages. Here, we describe (i) the dissociation of cells from whole Tg(mfap4:mCherry-F) zebrafish larvae using an enzyme-free and osmotically controlled buffer, (ii) the sorting of fluorescent macrophages by FACS and (iii) the preparation of high quality RNAs for meaningful gene expression analysis from a small number of isolated macrophages.•An optimized protocol in 5 steps to extract high quality RNAs from zebrafish macrophages.•A cell dissociation method using an enzyme-free and osmotically controlled buffer to prevent the alteration of macrophage activation status and limit cell mortality.•Production of high integrity RNAs from a small number of isolated macrophages.
    Keywords:  FACS-sorted; Macrophages dissociation; RNA quality; Zebrafish larva
    DOI:  https://doi.org/10.1016/j.mex.2022.101651
  12. Bio Protoc. 2022 Feb 20. 12(4): e4319
      Recent advancements in 3D microscopy have enabled scientists to monitor signals of multiple cells in various animals/organs. However, segmenting and tracking the moving cells in three-dimensional time-lapse images (3D + T images), to extract their dynamic positions and activities, remains a considerable bottleneck in the field. We developed a deep learning-based software pipeline called 3DeeCellTracker, which precisely tracks cells with large movements in 3D + T images, obtained from different animals or organs, using highly divergent optical systems. In this protocol, we explain how to set up the computational environment, the required data, and the procedures to segment and track cells with 3DeeCellTracker. Our protocol will help scientists to analyze cell activities/movements in 3D + T image datasets that have been difficult to analyze. Graphic abstract: The flowchart illustrating how to use 3DeeCellTracker. See the Equipment and Procedure sections for detailed explanations.
    Keywords:  3D microscopy; Cell segmentation; Cell tracking; Deep learning; Time lapse images
    DOI:  https://doi.org/10.21769/BioProtoc.4319
  13. Mitochondrion. 2022 Mar 25. pii: S1567-7249(22)00024-1. [Epub ahead of print]
      Mitochondrial permeability transition pore (mPTP) is a channel that opens at the inner mitochondrial membrane under conditions of stress. Sirtuin 3 (Sirt3) is a mitochondrial deacetylase known to play a major role in stress resistance and a regulatory role in cell death. This systematic review aims to elucidate the role of Sirt3 in mPTP inhibition. Electronic databases, including PubMed, EMBASE, Web of Science and Cochrane Library were searched up to May 2020. Original studies that investigated the relationship between Sirt3 and mPTP were included. Two reviewers independently extracted data on study characteristics, methods and outcomes. A total of 194 articles were found. Twenty-nine articles, which met criteria were included in the systematic review. Twenty-three studies provided evidence of the inhibitory effect of Sirt3 on the mPTP aperture. This review summarizes up-to-date evidence of the protective and inhibitory role of Sirt3 through deacetylating Cyclophilin D (CypD) on the mPTP aperture. Furthermore, we discuss the implications of this effect in disease.
    Keywords:  Cyclophilin D; Sirtuin3; deacetylase; mPTP; mitochondria
    DOI:  https://doi.org/10.1016/j.mito.2022.03.004
  14. J Mol Biol. 2022 Mar 24. pii: S0022-2836(22)00132-2. [Epub ahead of print] 167558
      Computational modeling of nucleic acids plays an important role in molecular biology, enhancing our general understanding of the relationship between structure and function. Biophysical studies have provided a wealth of information on how double-helical DNA responds to proteins and other molecules in its local environment but far less understanding of the larger scale structural responses found in protein-decorated loops and minicircles. Current computational models of DNA range from detailed all-atom molecular dynamics studies, which produce rich time and spatially dependent depictions of small DNA fragments, to coarse-grained simulations, which sacrifice detailed physical and chemical information to treat larger scale systems. The treatment of DNA used here, at the base-pair step level with rigid-body parameters, allows one to develop quality models hundreds of base pairs long from local, sequence-specific features found from experiment. The emDNA software takes advantage of this framework, producing elastically optimized models of DNA at thermal equilibrium with built-in or user-generated elastic models. This versatile program, in combination with case studies included in this article, allows users of any skill level to develop and investigate mesoscale models of their own design. The functionality of emDNA includes a tool to incorporate experiment-specific configurations, e.g. protein-bound and/or melted DNA from known high-resolution structures, within higher-order 3D models by fixing the orientation and position of user-specified base pairs. The software provides a new avenue into multiscale genetic modeling, giving a wide range of users a deeper understanding of DNA mesoscale organization and the opportunity to pose new questions in genetic research. The publicly available emDNA software, including build instructions and usage information, is available on GitHub (https://nicocvn.github.io/emDNA/).
    Keywords:  DNA loops; DNA minicircles; energy optimization; molecular modeling; protein-DNA interactions
    DOI:  https://doi.org/10.1016/j.jmb.2022.167558
  15. Reprod Fertil Dev. 2022 Apr 01.
       CONTEXT: While conventional semen analysis is a simple, time-saving, and economical means to evaluate sperm quality, it leaves biochemical and metabolic characteristics of spermatozoa aside. To address this issue, the use of fluorescent probes assessing functional sperm parameters, such as JC-1, DiOC6(3) and MitoTracker, has increased over the last decades. Apparently contradictory observations have nevertheless fostered an ongoing debate on their sensitivity and ability to evaluate the mitochondrial membrane potential (MMP) of sperm cells, thus warranting a re-examination of these probes.
    AIMS: The present study aims to elucidate the suitability and sensitivity of each probe to evaluate the MMP of bovine spermatozoa by flow cytometry.
    METHODS: Cryopreserved spermatozoa from ten bulls were thawed, stained with JC-1/SYTOXRed, DiOC6(3)/propidium iodide (PI) or MitoTracker Deep Red (MTDR)/PI, and evaluated with flow cytometry and fluorescence microscopy.
    KEY RESULTS: DiOC6(3), JC-1 and MTDR can be simultaneously co-stained with a viability marker. The results of the present study support the ability of DiOC6(3)/PI and JC-1/SYTOXRed, but not that of MTDR/PI, to monitor the MMP of spermatozoa.
    CONCLUSIONS: JC-1/SYTOXRed assessed by flow cytometry was found to be the most sensitive and robust fluorescent probe to assess MMP. Moreover, DiOC6(3)/PI could be a suitable alternative when the flow cytometer is not equipped with a red laser and/or an adequate optical filter.
    IMPLICATIONS: Both DiOC6(3) and JC-1, but not MTDR, could be used as probes to assess the mitochondrial membrane potential of bovine spermatozoa.
    DOI:  https://doi.org/10.1071/RD21355