Physiological and biochemical defects in functional interactions of mitochondrial DNA polymerase and DNA-binding mutants of single-stranded DNA-binding protein

Functional interactions between mitochondrial DNA polymerase (pol gamma) and mitochondrial single-stranded DNA-binding protein (mtSSB) from Drosophila embryos greatly enhance the overall activity of pol gamma by increasing primer recognition and binding and stimulating the rate of initiation of DNA strands (Farr, C. L., Wang, Y., and Kaguni, L. S. (1999) J. Biol. Chem. 274, 14779-14785). We show here that DNA-binding mutants of mtSSB are defective in stimulation of DNA synthesis by pol gamma. RNAi knock-down of mtSSB reduces expression to <5% of its normal level in Schneider cells, resulting in growth defects and in the depletion of mitochondrial DNA (mtDNA). Overexpression of mtSSB restores cell growth rate and the copy number of mtDNA, whereas overexpression of a DNA-binding and functionally impaired form of mtSSB neither rescues the cell growth defect nor the mtDNA depletion phenotype. Further development of Drosophila animal models, in which induced mtDNA depletion is manipulated by controlling exogenous expression of wild-type or mutant forms, will offer new insight into the mechanism and progression of human mtDNA depletion syndromes and possible intervention schemes.     

Depletion of mtDNA: syndromes and genes

Maintenance of mitochondrial DNA (mtDNA) requires the concerted activity of several nuclear-encoded factors that participate in its replication, being part of the mitochondrial replisome or ensuring the balanced supply of dNTPs to mitochondria. In the past decade, a growing number of syndromes associated with dysfunction due to tissue-specific depletion of mtDNA (MDS) have been reported. This article reviews the current knowledge of the genes responsible for these disorders, the impact of different mutations in the epidemiology of MDS and their role in the pathogenic mechanisms underlying the different clinical presentations.     

Liver mtDNA content increases during development: a comparison of methods and the importance of age- and tissue-specific controls for the diagnosis of mtDNA depletion

BACKGROUND: The quantitative loss of mitochondrial DNA (mtDNA) known as mtDNA depletion, often gives rise to liver disease. The diagnosis of mtDNA depletion syndrome is frequently imprecise, both for technical reasons and because of the lack of established age-adjusted normal ranges. We aimed to refine quantitative methods for diagnosing the hepatic type of mtDNA depletion syndrome, firstly by establishing an age-matched reference range for mitochondrial to nuclear DNA ratio (henceforth "mtDNA content") and secondly by investigating mtDNA in fibroblasts. METHODS: By comparing realtime PCR with an established method for quantifying mtDNA content we established a reference range for young children using biopsy and post-mortem material from patients <15 years. In addition, we investigated the arrangement of mtDNA in nucleoids from fibroblasts using fluorescence microscopy. RESULTS: Both methods showed that the mtDNA content of liver increases rapidly over the perinatal period. In a patient whose liver mtDNA content fell, but remained within the reference range, early investigation and age-matched controls were essential, as we found a progressive increase in muscle mtDNA copy number, respiratory chain activity and muscle power with age. In three further patients, fluorescence microscopy of the fibroblasts proved diagnostic. In one case a movement disorder was an important pointer. CONCLUSIONS: These cases highlight the (i) need for comparing mtDNA copy number data generated from patients to DNA isolated from an age-matched normal range from the tissue of interest and (ii) the utility of mtDNA staining with PicoGreen as a method to detect aberrant nucleoid morphology in mtDNA depletion patient fibroblast lines when affected tissues are not available for measuring mtDNA copy number.     

Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells

Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.     

Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number

Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.     

The conserved Mec1/Rad53 nuclear checkpoint pathway regulates mitochondrial DNA copy number in Saccharomyces cerevisiae

How mitochondrial DNA (mtDNA) copy number is determined and modulated according to cellular demands is largely unknown. Our previous investigations of the related DNA helicases Pif1p and Rrm3p uncovered a role for these factors and the conserved Mec1/Rad53 nuclear checkpoint pathway in mtDNA mutagenesis and stability in Saccharomyces cerevisiae. Here, we demonstrate another novel function of this pathway in the regulation of mtDNA copy number. Deletion of RRM3 or SML1, or overexpression of RNR1, which recapitulates Mec1/Rad53 pathway activation, resulted in an approximately twofold increase in mtDNA content relative to the corresponding wild-type yeast strains. In addition, deletion of RRM3 or SML1 fully rescued the approximately 50% depletion of mtDNA observed in a pif1 null strain. Furthermore, deletion of SML1 was shown to be epistatic to both a rad53 and an rrm3 null mutation, placing these three genes in the same genetic pathway of mtDNA copy number regulation. Finally, increased mtDNA copy number via the Mec1/Rad53 pathway could occur independently of Abf2p, an mtDNA-binding protein that, like its metazoan homologues, is implicated in mtDNA copy number control. Together, these results indicate that signaling through the Mec1/Rad53 pathway increases mtDNA copy number by altering deoxyribonucleoside triphosphate pools through the activity of ribonucleotide reductase. This comprises the first linkage of a conserved signaling pathway to the regulation of mitochondrial genome copy number and suggests that homologous pathways in humans may likewise regulate mtDNA content under physiological conditions.     

Inherited Mendelian defects of nuclear-mitochondrial communication affecting the stability of mitochondrial DNA

The presence of mtDNA abnormalities inherited as Mendelian traits indicates the existence of mutations in nuclear genes affecting the integrity of the mitochondrial genome. Two groups of nucleus-driven abnormalities have been described: qualitative alterations of mtDNA, i.e. multiple large-scale deletions of mtDNA, and quantitative decrease of the mtDNA copy number, i.e. tissue-specific depletion of mtDNA. Autosomal dominant or recessive (adPEO), progressive ophthalmoplegia and autosomal-recessive mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), are three neurodegenerative disorders associated with the coexistence of wild-type mtDNA with several deletion-containing mtDNA species. Heterozygous mutations of the genes encoding the muscle-heart isoform of the adenosine diphosphate/adenosine triphosphate mitochondrial translocator (ANT1), the main subunit of polymerase gamma (POLG1), and of the putative mtDNA helicase (Twinkle) have been found in adPEO families linked to three different loci, on chromosomes 4q34-35, 10q24, and 15q25, respectively. Mutations in the gene encoding thymidine phosphorylase have been identified in several MNGIE patients. Severe, tissue-specific depletion of mtDNA is the molecular hallmark of rapidly progressive hepatopathies or myopathies of infancy and childhood. Two genes, deoxyguanosine kinase and thymidine kinase type 2, both involved in the mitochondrion-specific salvage pathways of deoxynucleotide pools, have been associated with depletion syndromes in selected families.     

Autophagy determines mtDNA copy number dynamics during starvation

Derived from bacterial ancestors, mitochondria have maintained their own albeit strongly reduced genome, mitochondrial DNA (mtDNA), which encodes for a small and highly specialized set of genes. MtDNA exists in tens to thousands of copies packaged in numerous nucleoprotein complexes, termed nucleoids, distributed throughout the dynamic mitochondrial network. Our understanding of the mechanisms of how cells regulate the copy number of mitochondrial genomes has been limited. Here, we summarize and discuss our recent findings that Mip1/POLG (mitochondrial DNA polymerase gamma) critically controls mtDNA copy number by operating in 2 opposing modes, synthesis and, unexpectedly, degradation of mtDNA, when yeast cells face nutrient starvation. The balance of the 2 modes of Mip1/POLG and thus mtDNA copy number dynamics depends on the integrity of macroautophagy/autophagy, which sustains continuous synthesis and maintenance of mtDNA. In autophagy-deficient cells, a combination of nucleotide insufficiency and elevated mitochondrial ROS production impairs mtDNA synthesis and drives mtDNA degradation by the 3?-5?-exonuclease activity of Mip1/POLG resulting in mitochondrial genome depletion and irreversible respiratory deficiency.

Abbrivations: mtDNA: mitochondrial DNA; mtDCN: mitochondrial DNA copy number.     

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.     

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 regulation of mitochondrial DNA copy number in glioblastoma cells

As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions. It is not understood whether tumor-initiating cells regulate their mtDNA in a similar manner or whether mtDNA is essential for tumorigenesis. We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity. Differentiating multipotent glioblastoma cells failed to match the expansion in mtDNA copy number, patterns of gene expression and increased respiratory capacity observed in hNSCs. Partial depletion of glioblastoma cell mtDNA rescued mtDNA replication events and enhanced cell differentiation. However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH). The transfer of glioblastoma cells depleted to varying degrees of their mtDNA content into immunocompromised mice resulted in tumors requiring significantly longer to form compared with non-depleted cells. The number of tumors formed and the time to tumor formation was relative to the degree of mtDNA depletion. The tumors derived from mtDNA depleted glioblastoma cells recovered their mtDNA copy number as part of the tumor formation process. These outcomes demonstrate the importance of mtDNA to the initiation and maintenance of tumorigenesis in glioblastoma multiforme.     

Deoxyribonucleoside kinases in mitochondrial DNA depletion

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a heterogeneous group of mitochondrial disorders, manifested by a decreased mtDNA copy number and respiratory chain dysfunction. Primary MDS are inherited autosomally and may affect a single organ or multiple tissues. Mutated mitochondrial deoxyribonucleoside kinases; deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2), were associated with the hepatocerebral and myopathic forms of MDS respectively. dGK and TK2 are key enzymes in the mitochondrial nucleotide salvage pathway, providing the mitochondria with deoxyribonucleotides (dNP) essential for mtDNA synthesis. Although the mitochondrial dNP pool is physically separated from the cytosolic one, dNP's may still be imported through specific transport. Non-replicating tissues, where cytosolic dNP supply is down regulated, are thus particularly vulnerable to dGK and TK2 deficiency. The overlapping substrate specificity of deoxycytidine kinase (dCK) may explain the relative sparing of muscle in dGK deficiency, while low basal TK2 activity render this tissue susceptible to TK2 deficiency. The precise pathophysiological mechanisms of mtDNA depletion due to dGK and TK2 deficiencies remain to be determined, though recent findings confirm that it is attributed to imbalanced dNTP pools.     

Number matters: control of mammalian mitochondrial DNA copy number

Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA （mtDNA） depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration, diabetes, aging, and many other human diseases. Although it is known that the dynamics of the mammalian mitochondrial genome are not linked with that of the nuclear genome, very little is known about the mechanism of mtDNA propagation. Nevertheless, our understanding of the mode of mtDNA replication has ad- vanced in recent years, though not without some controversies. This review summarizes our current knowledge of mtDNA copy number control in mammalian cells, while focusing on both mtDNA replication and turnover. Although mtDNA copy number is seemingly in excess, we reason that mtDNA copy number control is an important aspect of mitochondrial genetics and biogenesis and is essential for normal cellular function.     

Reduced mitochondrial DNA copy number is correlated with tumor progression and prognosis in Chinese breast cancer patients

Somatic mutations and large-scale depletion in mitochondrial DNA (mtDNA) have been extensively detected in various human cancers. However, it still remains unclear whether the alterations in mtDNA content are related to the clinicopathological parameters and patient prognosis in breast cancer. In the present study, we analyzed the copy number of mtDNA in 59 cases of invasive breast tumors and paired nontumorous tissues using quantitative real-time PCR. Our data showed that the level of mtDNA was significantly decreased in tumor tissues as compared to the adjacent nontumorous counterparts (P = 0.001). The reduced copy number in mtDNA was associated with an older onset age (>or=50 years old, P = 0.035) as well as a higher histological grade (P = 0.012). Survival analysis measured by the Kaplan-Meier curves and the log-rank test indicated that patients with reduced mtDNA content had significantly poorer disease-free survival (DFS, P = 0.0079) and overall survival (OS, P = 0.011) rate. In addition, tumors harboring mutations in displacement (D)-loop region, particularly at the polycytidine stretch (T/N ratio = 64.3 +/- 8.2%) or close to the replication origins of the heavy-strand (T/N ratio = 68.7 +/- 5.5%), had a significantly lower copy number of mtDNA than the ones without D-loop alterations. Together, our results suggested that reduced copy number of mtDNA may be involved in breast neoplastic transformation or progression and mtDNA content might be potentially used as a tool to predict prognosis. Somatic mutation in the D-loop region probably is one of key contributing factors leading to decreased mtDNA level in breast tumors.     

Role of mitochondrial DNA replication during differentiation of reprogrammed stem cells

Mitochondrial DNA (mtDNA) is a 16.6 kb genome that encodes for 13 of the 100+ subunits of the electron transfer chain (ETC), whilst the other subunits are encoded by chromosomal DNA. The ETC is responsible for the generation of the majority of cellular ATP through the process of oxidative phosphorylation (OXPHOS). mtDNA is normally inherited from the population present in the mature oocyte just prior to fertilisation. However, following somatic cell nuclear transfer (SCNT), mtDNA can be transmitted from both the donor cell and the recipient oocyte. This heteroplasmic transmission of mtDNA is a random event and does not appear to be related to the amount of mtDNA contributed by the donor cell. The distribution of mtDNA is randomly segregated between blastomeres and differentiating tissues, and therefore the mtDNA complement transmitted to offspring tissue cannot be predicted. mtDNA divergence between the cytoplast and the donor cell in intra- and inter-specific crosses favours a slightly more diverse mtDNA haplotype. However, this is limited as interspecies SCNT (iSCNT) genetic divergence contributes to developmental failure. SCNT embryos demonstrate a plethora of aberrantly reprogrammed characteristics including the uncoordinated regulation of the mtDNA replication factors. This results in increased mtDNA copy number during preimplantation development and propagates the replication of donor cell mtDNA. These failures are likely to be a consequence of incompatible nuclear- and mtDNA -encoded proteins interacting within the ETC thus reducing ATP production. The outcomes would be similar to the severely debilitating or even fatal mtDNA diseases associated with genetic rearrangements to mtDNA or mtDNA depletion type syndromes and have serious implications for any form of karyoplast transfer approach. The only method to overcome the problems of heteroplasmy in SCNT embryos is to completely deplete the donor cell of its mtDNA prior to SCNT.     

Deoxyribonucleoside Kinases in Mitochondrial DNA Depletion

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a heterogeneous group of mitochondrial disorders, manifested by a decreased mtDNA copy number and respiratory chain dysfunction. Primary MDS are inherited autosomally and may affect a single organ or multiple tissues. Mutated mitochondrial deoxyribonucleoside kinases; deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2), were associated with the hepatocerebral and myopathic forms of MDS respectively. dGK and TK2 are key enzymes in the mitochondrial nucleotide salvage pathway, providing the mitochondria with deoxyribonucleotides (dNP) essential for mtDNA synthesis. Although the mitochondrial dNP pool is physically separated from the cytosolic one, dNP's may still be imported through specific transport. Non ‐replicating tissues, where cytosolic dNP supply is down regulated, are thus particularly vulnerable to dGK and TK2 deficiency. The overlapping substrate specificity of deoxycytidine kinase (dCK) may explain the relative sparing of muscle in dGK deficiency, while low basal TK2 activity render this tissue susceptible toTK2 deficiency. The precise patho‐physiological mechanisms of mtDNA depletion due to dGK and TK2 deficiencies remain to be determined, though recent findings confirm that it is attributed to imbalanced dNTP pools.