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.     

The FANCM family Mph1 helicase localizes to the mitochondria and contributes to mtDNA stability

Mitochondria are membrane-bound organelles found in eukaryotic cells where they generate energy through the respiratory chain. They contain their own genome that encodes genes critical to the mitochondrial function, but most of their protein content is synthetized from nuclear encoded genes. Damages to the mtDNA can cause mutations and rearrangements with an impact on the respiratory functions of the cell. DNA repair factors are able to localize to mitochondria to restore mtDNA integrity and ensure its proper inheritance. We describe in this article the mitochondrial localization of the Mph1/FANCM helicase that serves critical roles in nuclear DNA repair processes. Mph1 localizes to mitochondria and its functions contribute to the mtDNA integrity under mtDNA damaging conditions.     

Diseases caused by mutations in mitochondrial DNA

Mitochondrial diseases associated with mutations within mitochondrial genome are a subgroup of metabolic disorders since their common consequence is reduced metabolic efficiency caused by impaired oxidative phophorylation and shortage of ATP. Although the vast majority of mitochondrial proteins (approximately 1500) is encoded by nuclear genome, mtDNA encodes 11 subunits of respiratory chain complexes, 2 subunits of ATP synthase, 22 tRNAs and 2 rRNAs. Up to now, more than 250 pathogenic mutations have been described within mtDNA. The most common are point mutations in genes encoding mitochondrial tRNAs such as 3243A-->G and 8344T-->G that cause, respectively, MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) or MIDD (maternally-inherited diabetes and deafness) and MERRF (myoclonic epilepsy with ragged red fibres) syndromes. There have been also found mutations in genes encoding subunits of ATP synthase such as 8993T-->G substitution associated with NARP (neuropathy, ataxia and retinitis pigmentosa) syndrome. It is worth to note that mitochondrial dysfunction can also be caused by mutations within nuclear genes coding for mitochondrial proteins.     

The R336Q mutation in human mitochondrial EFTu prevents the formation of an active mt-EFTu·GTP·aa-tRNA ternary complex

The mitochondrial translational machinery allows the genes encoded by mitochondrial DNA (mtDNA) to be translated in situ. Mitochondrial translation requires a number of nucleus-encoded protein factors, some of which have been found to carry mutations in patients affected by mitochondrial encephalomyopathies. We have previously described the first, and so far only, mutation in the mitochondrial elongation factor Tu, mt-EFTu, in a baby girl with polycystic encephalopathy, micropolygyria, and leukodystrophic changes. Despite that the mutant mt-EFTu was present in normal amount in the patient's tissues, mitochondrial translation was severely reduced, determining multiple defects in the amount and activity of mtDNA-dependent respiratory chain complexes. By an in-vitro reconstructed translational system, we here provide evidence that the mutant mt-EFTu variant fails to bind to aminoacylated mitochondrial tRNAs, thus explaining the observed impairment of mitochondrial translation. This is the first analysis on the molecular mechanism of a mtDNA translation defect due to a nuclear gene mutation.     

Mitochondrial DNA mutations and breast tumorigenesis

Breast cancer is a heterogeneous disease and genetic factors play an important role in its genesis. Although mutations in tumor suppressors and oncogenes encoded by the nuclear genome are known to play a critical role in breast tumorigenesis, the contribution of the mitochondrial genome to this process is unclear. Like the nuclear genome, the mitochondrial genome also encodes proteins critical for mitochondrion functions such as oxidative phosphorylation (OXPHOS), which is known to be defective in cancer including breast cancer. Mitochondrial DNA (mtDNA) is more susceptible to mutations due to limited repair mechanisms compared to nuclear DNA (nDNA). Thus changes in mitochondrial genes could also contribute to the development of breast cancer. In this review we discuss mtDNA mutations that affect OXPHOS. Continuous acquisition of mtDNA mutations and selection of advantageous mutations ultimately leads to generation of cells that propagate uncontrollably to form tumors. Since irreversible damage to OXPHOS leads to a shift in energy metabolism towards enhanced aerobic glycolysis in most cancers, mutations in mtDNA represent an early event during breast tumorigenesis, and thus may serve as potential biomarkers for early detection and prognosis of breast cancer. Because mtDNA mutations lead to defective OXPHOS, development of agents that target OXPHOS will provide specificity for preventative and therapeutic agents against breast cancer with minimal toxicity.     

A whole mitochondrial genome screening in a MELAS patient: a novel mitochondrial tRNA(Val) mutation

Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA(Val). This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.     

Clinical and molecular findings in children with complex I deficiency

Isolated complex I deficiency, the most frequent OXPHOS disorder in infants and children, is genetically heterogeneous. Mutations have been found in seven mitochondrial DNA (mtDNA) and eight nuclear DNA encoded subunits, respectively, but in most of the cases the genetic basis of the biochemical defect is unknown. We analyzed the entire mtDNA and 11 nuclear encoded complex I subunits in 23 isolated complex I-deficient children, classified into five clinical groups: Leigh syndrome, progressive leukoencephalopathy, neonatal cardiomyopathy, severe infantile lactic acidosis, and a miscellaneous group of unspecified encephalomyopathies. A genetic definition was reached in eight patients (35%). Mutations in mtDNA were found in six out of eight children with Leigh syndrome, indicating a prevalent association between this phenotype and abnormalities in ND genes. In two patients with leukoencephalopathy, homozygous mutations were detected in two different nuclear-encoded complex I genes, including a novel transition in NDUFS1 subunit. In addition to these, a child affected by mitochondrial encephalomyopathy had heterozygous mutations in NDUFA8 and NDUFS2 genes, while another child with neonatal cardiomyopathy had a complex rearrangement in a single NDUFS7 allele. The latter cases suggest the possibility of unconventional patterns of inheritance in complex I defects.     

Genetic insights into OXPHOS defect and its role in cancer

Warburg proposed that cancer originates from irreversible injury to mitochondrial oxidative phosphorylation (mtOXPHOS), which leads to an increase rate of aerobic glycolysis in most cancers. However, despite several decades of research related to Warburg effect, very little is known about the underlying genetic cause(s) of mtOXPHOS impairment in cancers. Proteins that participate in mtOXPHOS are encoded by both mitochondrial DNA (mtDNA) as well as nuclear DNA. This review describes mutations in mtDNA and reduced mtDNA copy number, which contribute to OXPHOS defects in cancer cells. Maternally inherited mtDNA renders susceptibility to cancer, and mutation in the nuclear encoded genes causes defects in mtOXPHOS system. Mitochondria damage checkpoint (mitocheckpoint) induces epigenomic changes in the nucleus, which can reverse injury to OXPHOS. However, irreversible injury to OXPHOS can lead to persistent mitochondrial dysfunction inducing genetic instability in the nuclear genome. Together, we propose that "mitocheckpoint" led epigenomic and genomic changes must play a key role in reversible and irreversible injury to OXPHOS described by Warburg. These epigenetic and genetic changes underlie the Warburg phenotype, which contributes to the development of cancer.     

Disruption of Mitochondrion-To-Nucleus Interaction in Deceased Cloned Piglets

Most animals produced by somatic cell nuclear transfer (SCNT) are heteroplasmic for mitochondrial DNA (mtDNA). Oxidative phosphorylation (OXPHOS) in clones therefore requires the coordinated expression of genes encoded by the nuclear DNA and the two sources of mitochondria. Such interaction is rarely studied because most clones are generated using slaughterhouse oocytes of unrecorded origin. Here we traced the maternal lineages of seven diseased and five one-month-old live cloned piglets by sequencing their mtDNA. Additionally by using a 13K oligonucleotide microarray, we compared the expression profiles of nuclear and mtDNA-encoded genes that are involved in mitochondrial functions and regulation between the cloned groups and their age-matched controls (n=5 per group). We found that the oocytes used to generate the cloned piglets were of either the Large White or Duroc background, and oocyte genetic background was not related to the clones’ survival. Expression profiles of mtDNA-encoded genes in clones and controls showed intermixed clustering patterns without treatment or maternal lineage-dependency. In contrast, clones and controls clustered separately for their global and nuclear DNA-encoded mitochondrial genes in the lungs for both the deceased and live groups. Functional annotation of differentially expressed genes encoded by both nuclear and mtDNA revealed abnormal gene expression in the mitochondrial OXPHOS pathway in deceased clones. Among the nine differentially expressed genes of the OXPHOS pathway, seven were down-regulated in deceased clones compared to controls, suggesting deficiencies in mitochondrial functions. Together, these data demonstrate that the coordination of expression of mitochondrial genes encoded by nuclear and mtDNA is disrupted in the lung of diseased clones.     

Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast.

Instability of the mitochondrial genome (mtDNA) is a general problem from yeasts to humans. However, its genetic control is not well documented except in the yeast Saccharomyces cerevisiae. From the discovery, 50 years ago, of the petite mutants by Ephrussi and his coworkers, it has been shown that more than 100 nuclear genes directly or indirectly influence the fate of the rho(+) mtDNA. It is not surprising that mutations in genes involved in mtDNA metabolism (replication, repair, and recombination) can cause a complete loss of mtDNA (rho(0) petites) and/or lead to truncated forms (rho(-)) of this genome. However, most loss-of-function mutations which increase yeast mtDNA instability act indirectly: they lie in genes controlling functions as diverse as mitochondrial translation, ATP synthase, iron homeostasis, fatty acid metabolism, mitochondrial morphology, and so on. In a few cases it has been shown that gene overexpression increases the levels of petite mutants. Mutations in other genes are lethal in the absence of a functional mtDNA and thus convert this petite-positive yeast into a petite-negative form: petite cells cannot be recovered in these genetic contexts. Most of the data are explained if one assumes that the maintenance of the rho(+) genome depends on a centromere-like structure dispensable for the maintenance of rho(-) mtDNA and/or the function of mitochondrially encoded ATP synthase subunits, especially ATP6. In fact, the real challenge for the next 50 years will be to assemble the pieces of this puzzle by using yeast and to use complementary models, especially in strict aerobes.     

A whole mitochondrial genome screening in a MELAS patient: A novel mitochondrial tRNA mutation

Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA^Val. This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.     

Mitochondrial genome maintenance in health and disease

Human mitochondria harbor an essential, high copy number, 16,569 base pair, circular DNA genome that encodes 13 gene products required for electron transport and oxidative phosphorylation. Mutation of this genome can compromise cellular respiration, ultimately resulting in a variety of progressive metabolic diseases collectively known as ‘mitochondrial diseases’. Mutagenesis of mtDNA and the persistence of mtDNA mutations in cells and tissues is a complex topic, involving the interplay of DNA replication, DNA damage and repair, purifying selection, organelle dynamics, mitophagy, and aging. We briefly review these general elements that affect maintenance of mtDNA, and we focus on nuclear genes encoding the mtDNA replication machinery that can perturb the genetic integrity of the mitochondrial genome.     

Mitochondrial DNA variation is associated with measurable differences in life-history traits and mitochondrial metabolism in Drosophila simulans

Recent studies have used a variety of theoretical arguments to show that mitochondrial (mt) DNA rarely evolves as a strictly neutral marker and that selection operates on the mtDNA of many species. However, the vast majority of researchers are not convinced by these arguments because data linking mtDNA variation with phenotypic differences are limited. We investigated sequence variation in the three mtDNA and nine nuclear genes (including all isoforms) that encode the 12 subunits of cytochrome c oxidase of the electron transport chain in Drosophila. We then studied cytochrome c oxidase activity as a key aspect of mitochondrial bioenergetics and four life-history traits. In Drosophila simulans, sequence data from the three mtDNA encoded cytochrome c oxidase genes show that there are 76 synonymous and two nonsynonymous fixed differences among flies harboring siII compared with siIII mtDNA. In contrast, 13 nuclear encoded genes show no evidence of genetic subdivision associated with the mtDNA. Flies with siIII mtDNA had higher cytochrome c oxidase activity and were more starvation resistant. Flies harboring siII mtDNA had greater egg size and fecundity, and recovered faster from cold coma. These data are consistent with a causative role for mtDNA variation in these phenotypic differences, but we cannot completely rule out the involvement of nuclear genes. The results of this study have significant implications for the use of mtDNA as an assumed neutral marker and show that evolutionary shifts can involve changes in mtDNA despite the small number of genes encoded in the organelle genome.     

Mitochondrial involvement in Alzheimer's disease

The causes of most neurodegenerative diseases, including sporadic Alzheimer's disease (AD), remain enigmatic. There is, however, increasing evidence implicating mitochondrial dysfunction resulting from deafferentiation of disconnected neural circuits in the pathogenesis of energy deficit in AD. The patterns of reduced expression of both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) encoded genes is consistent with a physiological down-regulation of the mitochondrial respiratory chain in response to reduced neuronal activity. On the other hand, the role(s) of somatic cell or maternally inherited mtDNA mutations in the pathogenesis of mitochondrial dysfunction in AD are still controversial.     

Human diseases with impaired mitochondrial protein synthesis

Mitochondrial respiratory chain deficiencies represent one of the major causes of metabolic disorders that are related to genetic defects in mitochondrial or nuclear DNA. The mitochondrial protein synthesis allows the synthesis of the 13 respiratory chain subunits encoded by mtDNA. Altogether, about 100 different proteins are involved in the translation of the 13 proteins encoded by the mitochondrial genome emphasizing the considerable investment required to maintain mitochondrial genetic system. Mitochondrial protein synthesis deficiency can be caused by mutations in any component of the translation apparatus including tRNA, rRNA and proteins. Mutations in mitochondrial rRNA and tRNAs have been first identified in various forms of mitochondrial disorders. Moreover abnormal translation due to mutation in nuclear genes encoding tRNA-modifying enzymes, ribosomal proteins, aminoacyl-tRNA synthetases, elongation and termination factors and translational activators have been successively described. These deficiencies are characterized by a huge clinical and genetic heterogeneity hampering to establish genotype-phenotype correlations and an easy diagnosis. One can hypothesize that a new technique for gene identification, such as exome sequencing will rapidly allow to expand the list of genes involved in abnormal mitochondrial protein synthesis.     

Distinct nuclear gene expression profiles in cells with mtDNA depletion and homoplasmic A3243G mutation

The pathobiochemical pathways determining the wide variability in phenotypic expression of mitochondrial DNA (mtDNA) mutations are not well understood. Most pathogenic mtDNA mutations induce a general defect in mitochondrial respiration and thereby ATP synthesis. Yet phenotypic expression of the different mtDNA mutations shows large variations that are difficult to reconcile with ATP depletion as sole pathogenic factor, implying that additional mechanisms contribute to the phenotype. Here, we use DNA microarrays to identify changes in nuclear gene expression resulting from the presence of the A3243G diabetogenic mutation and from a depletion of mtDNA (rho0 cells). We find that cells respond mildly to these mitochondrial states with both general and specific changes in nuclear gene expression. This observation indicates that cells can sense the status of mtDNA. A number of genes show divergence in expression in rho0 cells compared to cells with the A3243G mutation, such as genes involved in oxidative phosphorylation. As a common response in A3243G and rho0 cells, mRNA levels for extracellular matrix genes are up-regulated, while the mRNA levels of genes involved in ubiquitin-mediated protein degradation and in ribosomal protein synthesis is down-regulated. This reduced expression is reflected at the level of cytosolic protein synthesis in both A3243G and rho0 cells. Our finding that mitochondrial dysfunction caused by different mutations affects nuclear gene expression in partially distinct ways suggests that multiple pathways link mitochondrial function to nuclear gene expression and contribute to the development of the different phenotypes in mitochondrial disease.     

线粒体疾病与核基因－线粒体基因的表达调控

线粒体与疾病是当前生物医学领域最前沿之一。本文简单介绍线粒体生物医学的基础知识、线粒体疾病的遗传模式,综述了近年来在线粒体DNA（mtDNA）突变和疾病、核基因突变和疾病等领域的研究进展,着重阐明核基因（特别是核修饰基因）调控mtDNA突变致病表达的分子机制。