Mitochondria: Biogenesis and mitophagy balance in segregation and clonal expansion of mitochondrial DNA mutations

Mitochondria are cytoplasmic organelles containing their own multi-copy genome. They are organized in a highly dynamic network, resulting from balance between fission and fusion, which maintains homeostasis of mitochondrial mass through mitochondrial biogenesis and mitophagy. Mitochondrial DNA (mtDNA) mutates much faster than nuclear DNA. In particular, mtDNA point mutations and deletions may occur somatically and accumulate with aging, coexisting with the wild type, a condition known as heteroplasmy. Under specific circumstances, clonal expansion of mutant mtDNA may occur within single cells, causing a wide range of severe human diseases when mutant overcomes wild type. Furthermore, mtDNA deletions accumulate and clonally expand as a consequence of deleterious mutations in nuclear genes involved in mtDNA replication and maintenance, as well as in mitochondrial fusion genes (mitofusin-2 and OPA1), possibly implicating mtDNA nucleoids segregation. We here discuss how the intricacies of mitochondrial homeostasis impinge on the intracellular propagation of mutant mtDNA.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

The human mitochondrial replication fork in health and disease

Mitochondria are organelles whose main function is to generate power by oxidative phosphorylation. Some of the essential genes required for this energy production are encoded by the mitochondrial genome, a small circular double stranded DNA molecule. Human mtDNA is replicated by a specialized machinery distinct from the nuclear replisome. Defects in the mitochondrial replication machinery can lead to loss of genetic information by deletion and/or depletion of the mtDNA, which subsequently may cause disturbed oxidative phosphorylation and neuromuscular symptoms in patients. We discuss here the different components of the mitochondrial replication machinery and their role in disease. We also review the mode of mammalian mtDNA replication.     

人类线粒体DNA异质性研究进展及其法医学应用

线粒体DNA(mitochondrial DNA mtDNA)的异质性自从被发现以来,一直被遗传学、进化学、发育遗传学以及法医遗传学、分子生物学领域所重视。由于线粒体异质性的存在,使得很多涉及疾病、进化、系统发育线粒体基因组与核基因组的相互作用关系、线粒体DNA复制机制以及法医学运用线粒体DNA进行实际案件评估的问题变得复杂化。此外线粒体DNA异质性的发生原因以及对线粒体异质性的检测方法标准化问题还没有一个统一的答案。针对线粒体DNA异质性带来的种种问题,近年来国内外取得了不少研究进展。     

Human mitochondrial DNA diseases and Drosophila models

Mutations that disrupt the mitochondrial genome cause a number of human diseases whose phenotypic presentation varies widely among tissues and individuals. This variability owes in part to the unconventional genetics of mitochondrial DNA(mtDNA), which includes polyploidy, maternal inheritance and dependence on nuclear-encoded factors. The recent development of genetic tools for manipulating mitochondrial genome in Drosophila melanogaster renders this powerful model organism an attractive alternative to mammalian systems for understanding mtDNA-related diseases. In this review, we summarize mtDNA genetics and human mtDNA-related diseases. We highlight existing Drosophila models of mtDNA mutations and discuss their potential use in advancing our knowledge of mitochondrial biology and in modeling human mitochondrial disorders. We also discuss the potential and present challenges of gene therapy for the future treatment of mtDNA diseases.     

Uncoupling the roles of the SUV3 helicase in maintenance of mitochondrial genome stability and RNA degradation

Yeast SUV3 is a nuclear encoded mitochondrial RNA helicase that complexes with an exoribonuclease, DSS1, to function as an RNA degradosome. Inactivation of SUV3 leads to mitochondrial dysfunctions, such as respiratory deficiency; accumulation of aberrant RNA species, including excised group I introns; and loss of mitochondrial DNA (mtDNA). Although intron toxicity has long been speculated to be the major reason for the observed phenotypes, direct evidence to support or refute this theory is lacking. Moreover, it remains unknown whether SUV3 plays a direct role in mtDNA maintenance independently of its degradosome activity. In this paper, we address these questions by employing an inducible knockdown system in Saccharomyces cerevisiae with either normal or intronless mtDNA background. Expressing mutants defective in ATPase (K245A) or RNA binding activities (V272L or ΔCC, which carries an 8-amino acid deletion at the C-terminal conserved region) resulted in not only respiratory deficiencies but also loss of mtDNA under normal mtDNA background. Surprisingly, V272L, but not other mutants, can rescue the said deficiencies under intronless background. These results provide genetic evidence supporting the notion that the functional requirements of SUV3 for degradosome activity and maintenance of mtDNA stability are separable. Furthermore, V272L mutants and wild-type SUV3 associated with an active mtDNA replication origin and facilitated mtDNA replication, whereas K245A and ΔCC failed to support mtDNA replication. These results indicate a direct role of SUV3 in maintaining mitochondrial genome stability that is independent of intron turnover but requires the intact ATPase activity and the CC conserved region.     

Replicating animal mitochondrial DNA

The field of mitochondrial DNA (mtDNA) replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s) used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark) has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading- and lagging-strand synthesis (resembling bacterial genome replication) and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS). The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase γ, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase). Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.     

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.     

Mitochondrial DNA replication and repair defects: Clinical phenotypes and therapeutic interventions

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.     

In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication

Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.     

Behavior of mitochondria and their nuclei during amoebae cell proliferation inPhysarum polycephalum

Summary We investigated the manner of mitochondrial DNA (mtDNA) replication and distribution during the culture ofPhysarum polycephalum amoebae cells by microphotometry, anti-BrdU immunofluorescence microscopy, and quantitative hybridization analysis. In amoebae cells ofP. polycephalum, the number of mitochondria per cell and the shape of both mitochondria and mitochondrial nuclei (mt-nuclei) noticeably changed over the culture period. At the time of transfer, about 27 short ellipsoidal shaped mitochondria, which each contained a small amount of DNA, were observed in each cell. The number of mitochondria per cell decreased gradually, while the amount of mtDNA in an mt-nucleus and the length of mt-nuclei increased gradually. Midway through the middle logarithmic growth phase, the number of mitochondria per cell reached a minimum (about 10 mitochondria per cell), but most mtnuclei assumed an elongated shape and contained a large amount of mtDNA. During the late log- and stationary-growth phase, the number of mitochondria per cell increased gradually, while the amount of DNA in an mt-nucleus and mt-nuclei length decreased gradually. Upon completion of the stationary phase, the number and condition of mitochondria within cells returned to that first observed at the time of transfer. The total amount of mtDNA in a cell increased about 1.6-fold the first day, decreased immediately, then maintained a constant level ranging from 130 to 160 T. Except for the fact that mtDNA synthesis began earlier than synthesis of cell nuclei, the rate of increase in mtDNA paralleled that of cell-nuclear DNA throughout the culture. These results indicate that mtDNA is continuously replicated in pace with cell proliferation and the rate of mitochondrial division varies during culture; this mitochondrial division does not synchronize with either mtDNA replication or cell division. Furthermore, we observed the spatial distribution of DNA replication sites along mt-nuclei. Replication began at several sites scattered along an mt-nucleus, and the number of replication sites increased as the length of mt-nuclei increased. These results indicate that mtDNA replication progresses in adjacent replicons, which are collectively termed a mitochondrial replicon cluster.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system - BrdU 5-bromodeoxyuridine - FITC fluorescein isothiocyanate     

Inherited mitochondrial variants are not a major cause of age-related hearing impairment in the European population

Mitochondrial DNA (mtDNA) mutations have been implicated in various age-related diseases. To further clarify the role of mtDNA variants in age-related hearing impairment (ARHI), we determined the DNA sequence of the entire mitochondrial genome of 400 individuals using the Affymetrix Human Mitochondrial Resequencing Array. These were the 200 worst hearing and the 200 best hearing from a collection of 947 Belgian samples. We performed association tests with individual mitochondrial variants, comparison of the mutation load, and association with European haplogroups and their interaction with environmental risk factors. We also tested the influence of rare variants on ARHI. None of these tests showed any association with ARHI.     

Reduced stimulation of recombinant DNA polymerase γ and mitochondrial DNA (mtDNA) helicase by variants of mitochondrial single-stranded DNA-binding protein (mtSSB) correlates with defects in mtDNA replication in animal cells

The mitochondrial single-stranded DNA-binding protein (mtSSB) is believed to coordinate the functions of DNA polymerase γ (pol γ) and the mitochondrial DNA (mtDNA) helicase at the mtDNA replication fork. We generated five variants of the human mtSSB bearing mutations in amino acid residues specific to metazoans that map on the protein surface, removed from the single-stranded DNA (ssDNA) binding groove. Although the mtSSB variants bound ssDNA with only slightly different affinities, they exhibited distinct capacities to stimulate the DNA polymerase activity of human pol γ and the DNA unwinding activity of human mtDNA helicase in vitro. Interestingly, we observed that the variants with defects in stimulating pol γ had unaltered capacities to stimulate the mtDNA helicase; at the same time, variants showing reduced stimulation of the mtDNA helicase activity promoted DNA synthesis by pol γ similarly to the wild-type mtSSB. The overexpression of the equivalent variants of Drosophila melanogaster mtSSB in S2 cells in culture caused mtDNA depletion under conditions of mitochondrial homeostasis. Furthermore, we observed more severe reduction of mtDNA copy number upon expression of these proteins during recovery from treatment with ethidium bromide, when mtDNA replication is stimulated in vivo. Our findings suggest that mtSSB uses distinct structural elements to interact functionally with its mtDNA replisome partners and to promote proper mtDNA replication in animal cells.     

Multifactorial nature of high frequency of mitochondrial DNA mutations in somatic mammalian cells

The high frequency of mitochondrial DNA (mtDNA) mutations in somatic mammalian cells, which is more than two orders of magnitude higher than the mutation frequency of nuclear DNA (nDNA), significantly correlates with development of a variety of mitochondrial diseases (neurodegenerative diseases, cardiomyopathies, type II diabetes mellitus, cancer, etc.). A direct cause—consequence relationship has been established between mtDNA mutations and aging phenotypes in mammals. However, the unclear nature of the high frequency of mtDNA mutations requires a comprehensive consideration of factors that contribute to this phenomenon: oxidative stress, features of structural organization and repair of the mitochondrial genome, ribonucleotide reductase activity, replication errors, mutations of nuclear genes encoding mitochondrial proteins.     

Discovery of a Novel Function for Human Rad51: MAINTENANCE OF THE MITOCHONDRIAL GENOME*

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free repair of DNA double strand breaks. Rad51 is the central catalyst of HR in all eukaryotes, and to this point studies of human Rad51 have focused exclusively on events occurring within the nucleus. However, substantial amounts of HR proteins exist in the cytoplasm, yet the function of these protein pools has not been addressed. Here, we provide the first demonstration that Rad51 and the related HR proteins Rad51C and Xrcc3 exist in human mitochondria. We show stress-induced increases in both the mitochondrial levels of each protein and, importantly, the physical interaction between Rad51 and mitochondrial DNA (mtDNA). Depletion of Rad51, Rad51C, or Xrcc3 results in a dramatic decrease in mtDNA copy number as well as the complete suppression of a characteristic oxidative stress-induced copy number increase. Our results identify human mtDNA as a novel Rad51 substrate and reveal an important role for HR proteins in the maintenance of the human mitochondrial genome.     

Mitochondrial DNA recombination in Brassica campestris

The structure of the mitochondrial genome in plants is unclear, but appears to consist of mostly linear DNA with some other structures, including branched molecules and subgenomic circles. Mitochondrial DNA (mtDNA) recombination was analyzed in Brassica campestris, which has one of the smallest mitochondrial genomes (218 kb) in higher plants. Field-inversion gel electrophoresis (FIGE) separated mtDNA into discrete populations that each represents the entire genome. Electron microscopy revealed large, mostly linear molecules trapped in the wells, slower migrating populations with mostly linear DNA and a low level of circular and networked mtDNA molecules of 10–140 kbp, and a fast migrating population of 10–50 kbp linear mtDNA. Some smaller than genome size circular molecules and circles with tails were observed, and may represent recombination or rolling circle replication intermediates. Hybridization of end-labeled mtDNA suggests there may be specific ends (or recombination hotspots) for some linear molecules. Analysis of mtDNA enriched by BND-cellulose and separated by two-dimensional agarose gel electrophoresis shows the presence of complex recombination structures and the presence of significant single-stranded regions in mtDNA. These findings provide further evidence that DNA recombination contributes to the complex structure of mtDNA in plants.     

The landscape of mitochondrial DNA variation in human colorectal cancer on the background of phylogenetic knowledge

Recently, an increasing number of studies indicate that mutations in mitochondrial genome may contribute to cancer development or metastasis. Hence, it is important to determine whether the mitochondrial DNA might be a good, clinically applicable marker of cancer. This review describes hereditary as well as somatic mutations reported in mitochondrial DNA of colorectal cancer cells. We showed here that the entire mitochondrial genome mutational spectra are different in colorectal cancer and non-tumor cells. We also placed the described mutations on the phylogenetic context, which highlighted the recurrent problem of data quality. Therefore, the most important rules for adequately assessing the quality of mitochondrial DNA sequence analysis in cancer have been summarized. As follows from this review, neither the reliable spectrum of mtDNA somatic mutations nor the association between hereditary mutations and colorectal cancer risk have been resolved. This indicates that only high resolution studies on mtDNA variability, followed by a proper data interpretation employing phylogenetic knowledge may finally verify the utility of mtDNA sequence (if any) in clinical practice.     

Defects in mitochondrial DNA replication and human disease

Mitochondrial DNA (mtDNA) is replicated by the DNA polymerase g in concert with accessory proteins such as the mtDNA helicase, single stranded DNA binding protein, topoisomerase, and initiating factors. Nucleotide precursors for mtDNA replication arise from the mitochondrial salvage pathway originating from transport of nucleosides, or alternatively from cytoplasmic reduction of ribonucleotides. Defects in mtDNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mtDNA deletions, point mutations, or depletion which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mtDNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders, such as progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). This review focuses on our current knowledge of genetic defects of mtDNA replication (POLG, POLG2, C10orf2) and nucleotide metabolism (TYMP, TK2, DGOUK, and RRM2B) that cause instability of mtDNA and mitochondrial disease.     

The mitochondrial DNA helicase TWINKLE can assemble on a closed circular template and support initiation of DNA synthesis

Mitochondrial DNA replication is performed by a simple machinery, containing the TWINKLE DNA helicase, a single-stranded DNA-binding protein, and the mitochondrial DNA polymerase γ. In addition, mitochondrial RNA polymerase is required for primer formation at the origins of DNA replication. TWINKLE adopts a hexameric ring-shaped structure that must load on the closed circular mtDNA genome. In other systems, a specialized helicase loader often facilitates helicase loading. We here demonstrate that TWINKLE can function without a specialized loader. We also show that the mitochondrial replication machinery can assemble on a closed circular DNA template and efficiently elongate a DNA primer in a manner that closely resembles initiation of mtDNA synthesis in vivo.