In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells

The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. The D198A amino acid replacement abolished detectable 3'-5' (proofreading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous culture. Further culture resulted in the selection of cells with an inactivated mutator polymerase, and a reduced mutation load in mtDNA. Transient expression of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial DNA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression. These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities.     

Increased sensitivity to cis-diamminedichloroplatinum induced apoptosis with mitochondrial DNA depletion

Malignant cells harbor mechanisms which allow escape from drug-induced apoptosis, and the drug-resistance phenotype can be significantly associated with resistance to programmed cell death. There is accumulating evidence that mitochondria play a role in the tumorigenic phenotype, including the relative resistance to apoptosis. Whether changes at the mitochondrial level per se, would impact on the relative sensitivity of malignant cells to undergo drug-induced apoptosis, is not know. Accordingly, we determined if depleting mitochondrial DNA (mtDNA) would change the susceptibility of U937 cells to undergo apoptosis. With depletion, increases in sensitivity to cis-diamminedichoroplatinum (cisplatin)-induced apoptosis was observed. This sensitivity could be reverted to the parental phenotype by transforming the depleted cells with normal platelet mitochondria. mRNA expression of BAX, BCL2, MDR1, MRP, ERCC1 and ERCC2, putatively associated with cisplatin resistance to apoptotic death was unchanged. Inhibition of mitochondrial ATP production by oligomycin did not result in a change in ATP levels, indicating energetics were not playing a role in the observed phenotype changes. All U937 cells (with/without mtDNA) continued to respond to cisplatin by an apoptotic death. MtDNA-encoded molecules may be playing a role in the relative sensitivity of cells to undergo a cisplatin-induced apoptotic death, but may not be required for cells to undergo apoptosis per se.     

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.     

Maternal inheritance of mitochondrial DNA polymorphisms in cultured human fibroblasts

We have isolated the total cellular DNA from the cultured diploid fibroblasts of a six-member, three-generation human family. Using a specific radioactive probe for mitochondrial (mt) sequences we have identified new polymorphic variants in this family for the Hhal restriction endonuclease cleavage pattern of the mtDNA. The inheritance of these cleavage patterns verifies the maternal inheritance of mtDNA through all three generations.     

Deleterious mitochondrial DNA mutations accumulate in aging human tissues.

This paper reviews the current state of knowledge of the contribution of mitochondrial DNA (mtDNA) mutations to the phenotype of aging. Its major focus is on the discovery of deletions of mtDNA which previously were thought to occur only in individuals with neuromuscular disease. One particular deletion (mtDNA4977) accumulates with age primarily in non-dividing cells such as muscle and brain of normal individuals. The level of the deletion rises with age by more than 1000 fold in heart and brain and to a lesser extent in other tissues. In the brain, different regions have substantially different levels of the deletion. High levels of accumulation of the deletion in tissues are correlated with high oxygen consumption. We speculate that oxidative damage to mtDNA may be 'catastrophic'; mutations affecting mitochondrially encoded polypeptides involved in electron transport could increase free radical generation leading to more mtDNA damage.     

Heteroplasmic mutation of mitochondrial DNA D-loop and 4977-bp deletion in human cancer cells during mitochondrial DNA depletion

Somatic mutations in mitochondrial DNA (mtDNA) have been demonstrated in various human cancers. Many cancers have high frequently of mtDNA with homoplasmic point mutations, and carry less frequently of mtDNA with large-scale deletions as compared with corresponding non-cancerous tissue. Moreover, most cancers harbor a decreased copy number of mtDNA than their corresponding non-cancerous tissue. However, it is unclear whether the process of decreasing in mtDNA content would be involved in an increase in the heteroplasmic level of somatic mtDNA point mutation, and/or involved in a decrease in the proportion of mtDNA with large-scale deletion in cancer cells. In this study, we provided evidence that the heteroplasmic levels of variations in cytidine number in np 303-309 poly C tract of mtDNA in three colon cancer cells were not changed during an ethidium bromide-induced mtDNA depleting process. In the mtDNA depleting process, the proportions of mtDNA with 4977-bp deletion in cybrid cells were not significantly altered. These results suggest that the decreasing process of mtDNA copy number per se may neither contribute to the shift of homoplasmic/heteroplasmic state of point mutation in mtDNA nor to the decrease in proportion of mtDNA with large-scale deletions in cancer cells. Mitochondrial genome instability and reduced mtDNA copy number may independently occur in human cancer.     

Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining

Human mitochondria DNA (mtDNA) is arranged within the mitochondria into discrete DNA-protein complexes, termed nucleoids. The size of the human mitochondrial genome is less than that of yeast and is more difficult to visualise by fluorescent DNA stains such as DAPI and Hoescht. We have developed a simple yet effective method to visualise mtDNA in situ within living cells using the fluorescent stain PicoGreen. Quantitative analysis shows that PicoGreen can be used to estimate the degree of mtDNA depletion within living cells. We have used this approach to study the arrangement and fluorescence of nucleoids in cells depleted of mtDNA by treatment with the anti-viral nucleoside analogue, 2',3'-dideoxycytidine. We also studied the distribution of mtDNA in fibroblasts cultured from patients with mitochondrial disease. Combining PicoGreen staining with histochemical and immunocytochemical approaches enabled us to examine the effects of mtDNA depletion on mtDNA-related components at the level of single cells. This method is able to detect an intermediate degree of mtDNA depletion in living cells, and can be used to detect mtDNA free cells (rho0 cells) in culture even at very low numbers. We have also adapted the technique to efficiently sort rho0 cells from populations of normal cells by fluorescent-assisted cell sorting (FACS), without the need for selection of respiratory competence. This should be useful for the construction of new trans-mitochondrial 'cybrid' cell lines.     

Mutations in DNA polymerase gamma cause error prone DNA synthesis in human mitochondrial disorders

This paper summarizes recent advances in understanding the links between the cell's ability to maintain integrity of its mitochondrial genome and mitochondrial genetic diseases. Human mitochondrial DNA is replicated by the two-subunit DNA polymerase gamma (polgamma). We investigated the fidelity of DNA replication by polgamma with and without exonucleolytic proofreading and its p55 accessory subunit. Polgamma has high base substitution fidelity due to efficient base selection and exonucleolytic proofreading, but low frameshift fidelity when copying homopolymeric sequences longer than four nucleotides. Progressive external ophthalmoplegia (PEO) is a rare disease characterized by the accumulation of large deletions in mitochondrial DNA. Recently, several mutations in the polymerase and exonuclease domains of the human polgamma have been shown to be associated with PEO. We are analyzing the effect of these mutations on the human polgamma enzyme. In particular, three autosomal dominant mutations alter amino acids located within polymerase motif B of polgamma. These residues are highly conserved among family A DNA polymerases, which include T7 DNA polymerase and E.coli pol I. These PEO mutations have been generated in polgamma to analyze their effects on overall polymerase function as well as the effects on the fidelity of DNA synthesis. One mutation in particular, Y955C, was found in several families throughout Europe, including one Belgian family and five unrelated Italian families. The Y955C mutant polgamma retains a wild-type catalytic rate but suffers a 45-fold decrease in apparent binding affinity for the incoming dNTP. The Y955C derivative is also much less accurate than is wild-type polgamma, with error rates for certain mismatches elevated by 10- to 100-fold. The error prone DNA synthesis observed for the Y955C polgamma is consistent with the accumulation of mtDNA mutations in patients with PEO. The effects of other polgamma mutations associated with PEO are discussed.     

Age-associated oxygen damage and mutations in mitochondrial DNA in human hearts.

Some mutations in mitochondrial DNA (mtDNA) causing a number of neuromuscular diseases are suggested to arise spontaneously during the life of an individual. To substantiate the extent and the rate of these somatic mutations, mtDNA specimens from post-mortem human heart muscles of subjects in differing age groups were hydrolyzed. 8-Hydroxy-deoxyguanosine (8-OH-dG), a hydroxyl-radical adduct of deoxyguanosine, in mtDNA, was quantitatively determined using a micro high-performance liquid chromatography/mass spectrometry system. In each specimen, the mtDNA with a 7.4 kilo base-pair deletion was quantified by the kinetic polymerase chain reaction method. In association with age, the 8-OH-dG content accumulated exponentially up to 1.5% with a correlative increase in the content of the deleted mtDNA up to 7%. Clear correlation between the 8-OH-dG content in mtDNA and the population of mtDNA with a deletion (r = 0.93, P < 0.01) gives insight into the mechanism for the generation of a large deletion. These results indicate that accumulation of somatically acquired oxygen damage together with age-associated mutations in mtDNA which lead to bioenergetic deficiency and the heart muscle weakness are inevitable in human life.     

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.     

Origins of human mitochondrial point mutations as DNA polymerase gamma-mediated errors

Mitochondrial mutational spectra in human cells, tissues and derived tumors for bp 10,030-10,130 are essentially identical, suggesting a predominant mutagenic role for endogenous processes. We hypothesized that errors mediated by mitochondrial DNA polymerase gamma were the primary sources of mutations. Point mutations created in this sequence by human DNA pol gamma in vitro were thus compared to the eighteen mutational hotspots, all single base substitutions, previously found in human tissues. The set of concordant hotspots accounted for 83% of these in vivo mutational events. About half of these mutations are insensitive to prolonged heating of DNA during PCR and half increase proportionally with heating time at 98 degrees C. Primary misincorporation errors and miscopying errors past thermal denaturing products such as deaminated cytosines (uracils) thus appear to be of approximately equal importance. For the sequence studied, these data support the conclusion that, endogenous error mediated by DNA pol gamma constitutes the primary source of mitochondrial point mutations in human tissues.     

Coenzyme Q10 deficiency in mitochondrial DNA depletion syndromes

We evaluated coenzyme Q₁₀ (CoQ) levels in patients studied under suspicion of mitochondrial DNA depletion syndromes (MDS) (n = 39). CoQ levels were quantified by HPLC, and the percentage of mtDNA depletion by quantitative real-time PCR. A high percentage of MDS patients presented with CoQ deficiency as compared to other mitochondrial patients (Mann–Whitney-U test: p = 0.001). Our findings suggest that MDS are frequently associated with CoQ deficiency, as a possible secondary consequence of disease pathophysiology. Assessment of muscle CoQ status seems advisable in MDS patients since the possibility of CoQ supplementation may then be considered as a candidate therapy.     

The investigation and diagnosis of pathogenic mitochondrial DNA mutations in human urothelial cells

Patients with mitochondrial DNA disease are amongst the most challenging to diagnose and manage given the striking phenotypic and genetic heterogeneity, which characterise these conditions. Recently, we and others have demonstrated the m.3243A>G mutation, one of the most common mitochondrial DNA pathogenic mutations, is present at clinically relevant levels in urinary epithelium, thus providing a practical, non-invasive test for diagnosis and mutation screening. In this study we further evaluate the use of these cells in detecting the m.3243A>G mutation, other mtDNA tRNA gene point mutations including the m.8344A>G mutation and single large-scale mtDNA deletions. We observe a robust relationship between m.3243A>G levels in urothelial cells and clinically affected tissues that does not change with time. Conversely, single large-scale mtDNA deletions can be detected in urothelial cells, with higher levels present in younger patients with more severe disease, but generally mtDNA deletion levels are not representative of those seen in a clinically affected tissue. Our results have implications for the diagnosis, management and counselling of families with mtDNA disease.     

Kinetics of ultraviolet induced DNA excision repair in rat and human fibroblasts

To obtain more information on the well-documented low excision-repair capacity of rodent cells in comparison with human cells, we have studied this form of DNA repair in UV-irradiated human and rat skin fibroblasts. For this purpose, we have determined (i) unscheduled DNA synthesis (UDS), using autoradiography, (ii) the number and size of repaired sites with the bromodeoxyuridine (BrdU) photolysis assay and (iii) the removal of Micrococcus luteus UV-endonuclease susceptible sites (ESS). We found rat cells to be quite capable of performing DNA-repair synthesis, as demonstrated by both UDS and BrdU photolysis, whereas they almost completely lacked the capacity to remove pyrimidine dimers, as indicated by the persistence of ESS. This discrepancy will be discussed in terms of the types of mechanisms by which mammalian cells may recognize and remove UV-induced photoproducts.     

Calcium store depletion induced by mitochondrial uncoupling in prostatic cells

The effects of mitochondrial uncoupling on the calcium homeostasis of prostatic cells were investigated using the prostatic cancer cell line LNCaP and indo-1 spectrofluorimetry. Carbonyl cyanide m-chloro-phenylhydrazone (CCCP) was used as uncoupler. Resting LNCaP cells responded to CCCP by a biphasic increase in [Ca2+]i. The first phase of increase which corresponded to the release of a mitochondrial CCCP-sensitive Ca2+ store was followed by a second increase phase consisting of Ca2+ influx through the plasma membrane. The relationship between the CCCP- and the InsP3-sensitive stores was investigated using thapsigargin (TG). The release part of the Ca2+ response to TG was reduced in a time-dependent manner by previous exposure of the cells to CCCP, suggesting that CCCP also acts on non-mitochondrial stores. Our results show that CCCP releases Ca2+ from both mitochondrial and non-mitochondrial stores in prostatic cells. The possible mechanisms of these effects are discussed.     

Unscheduled synthesis of DNA and poly(ADP-ribose) in human fibroblasts following DNA damage

Unscheduled DNA synthesis has been measured in human fibroblasts under conditons of reduced rates of conversion of NAD to poly(ADP-ribose). Cells heterozygous for the xeroderma pigmentosum genotype showed normal rates of UV induced unscheduled DNA synthesis under conditions in which the rate of poly(ADP-ribose) synthesis was one-half the rate of normal cells. The addition of theophylline, a potent inhibitor of poly(ADP-ribose) polymerase, to the culture medium of normal cells blocked over 90% of the conversion of NAD to poly(ADP-ribose) following treatment with UV or N-methyl-N′-nitro-N-nitro-soguanidine but did not affect the rate of unscheduled DNA synthesis.     

ATM-mediated mitochondrial damage response triggered by nuclear DNA damage in normal human lung fibroblasts

Ionizing radiation (IR) elevates mitochondrial oxidative phosphorylation (OXPHOS) in response to the energy requirement for DNA damage responses. Reactive oxygen species (ROS) released during mitochondrial OXPHOS may cause oxidative damage to mitochondria in irradiated cells. In this paper, we investigated the association between nuclear DNA damage and mitochondrial damage following IR in normal human lung fibroblasts. In contrast to low-doses of acute single radiation, continuous exposure of chronic radiation or long-term exposure of fractionated radiation (FR) induced persistent Rad51 and γ-H2AX foci at least 24 hours after IR in irradiated cells. Additionally, long-term FR increased mitochondrial ROS accompanied with enhanced mitochondrial membrane potential (ΔΨm) and mitochondrial complex IV (cytochrome c oxidase) activity. Mitochondrial ROS released from the respiratory chain complex I caused oxidative damage to mitochondria. Inhibition of ATM kinase or ATM loss eliminated nuclear DNA damage recognition and mitochondrial radiation responses. Consequently, nuclear DNA damage activates ATM which in turn increases ROS level and subsequently induces mitochondrial damage in irradiated cells.

In conclusion, we demonstrated that ATM is essential in the mitochondrial radiation responses in irradiated cells. We further demonstrated that ATM is involved in signal transduction from nucleus to the mitochondria in response to IR.     

The distribution of DNA excision-repair sites in human diploid fibroblasts following ultraviolet irradiation

Using the technique for separating DNA fragments containing excision-repair sites from total genomic DNA as described in the previous paper (Cohn, S. M., and Lieberman, M. W. (1984) J. Biol. Chem. 259, 12456-12462), we have developed a method for directly determining the distribution of excision-repair sites in the genome. DNA was prepared from confluent, diploid human fibroblasts which had been irradiated with ultraviolet light and incubated in the presence of 5-bromo-2'-deoxyuridine (BrdUrd), repaired fragments were isolated, and the dependence of the fraction of total DNA fragments containing excision-repair sites on DNA fragment length was determined by electrophoretic analysis. The observed dependence was compared to the relationship expected for a random distribution of repair sites. At 36 h following 3 J/m2 UV, the distribution of repair sites was indistinguishable from a random distribution; however, at doses of UV above 6 J/m2, the observed dependence indicated that the distribution of repair sites was nonrandom. A time course of the distribution of repair sites following 12 J/m2 UV was clearly nonrandom from 4 h after irradiation until at least 36 h following irradiation. By 72 h, however, the distribution had become random. In cells treated with hydroxyurea, a reduced number of excision-repair sites were present, but the distribution of repair sites was also nonrandom. Autoradiographic analysis of the amount of unscheduled DNA synthesis in individual nuclei suggested that the nonrandom distribution of repair sites did not result from variable extents of repair synthesis in different cell populations or from cell death.