Mitochondrial Replication Research Articles

Mitochondrial mutations, cellular instability and ageing: modelling the population dynamics of mitochondria.

All eukaryotic cells rely on mitochondrial respiration as their major source of metabolic energy (ATP). However, the mitochondria are also the main cellular source of oxygen radicals and the mutation rate of mtDNA is much higher than for chromosomal DNA. Damage to mtDNA is of great importance because it will often impair cellular energy production. However, damaged mitochondria can still replicate because the enzymes for mitochondrial replication are encoded entirely in the cell nucleus. For these reasons, it has been suggested that accumulation of defective mitochondria may be an important contributor to loss of cellular homoeostasis underlying the ageing process. We describe a mathematical model which treats the dynamics of a population of mitochondria subject to radical-induced DNA mutations. The model confirms the existence of an upper threshold level for mutations beyond which the mitochondrial population collapses. This threshold depends strongly on the division rate of the mitochondria. The model also reproduces and explains (i) the decrease in mitochondrial population with age, (ii) the increase in the fraction of damaged mitochondria in old cells, (iii) the increase in radical production per mitochondrion, and (iv) the decrease in ATP production per mitochondrion.

Transcribed heteroplasmic repeated sequences in the porcine mitochondrial DNA D-loop region.

The mitochondrial D-loop region of the pig, Sus scrofa, was found to be several hundred base pairs larger than the corresponding region in cow, a related artiodactyl species, primarily because of an insertion containing the tandemly repeated sequence CGTGCGTACA. Porcine mitochondrial DNA from the tissue of a single animal exhibits a large population of length polymorphs, each member of which may have as few as 14 or as many as 29 of these repeat units. This intracellular variability may be due to the repeated and self-complementary properties of this sequence, which would favor mispairing and lead to replication slippage. The repeat domain is unusual in that symmetry properties suggest it may assume alternative conformations including cruciforms and left-handed (Z) DNA. It also appears to be the longest known, naturally occurring, alternating purine-pyrimidine sequence. In order to understand the functional significance of this heteroplasmic domain that potentially disrupts a key regulatory region in the mitochondrial genome, RNA and DNA mapping studies were conducted which located this region between the H-strand replication origin and the putative L-strand transcriptional start site. H-strand RNA analysis demonstrated that this heteroplasmic region is transcribed and, therefore, that priming for H-strand DNA replication in mitochondria is independent of the primer RNA length or secondary structure.

RIM2, MSI1 and PGI1 are located within an 8 kb segment of Saccharomyces cerevisiae chromosome II, which also contains the putative ribosomal gene L21 and a new putative essential gene with a leucine zipper motif.

We report the DNA sequence of an 8 kb segment localized on the right arm of chromosome II from Saccharomyces cerevisiae. The sequence reveals the presence of eight open reading frames (ORFs). Three of them, YBR1402, YBR1405 and YBR1406 are previously sequenced genes, respectively the RIM2 (replication in mitochondria), MSI1 (multicopy suppressor of IRA1 gene) and PGI1 (phosphoglucoisomerase) genes. The predicted product of the ORF YBR1401 could be the putative yeast ribosomal protein L21. A new essential gene, YBR1403, has been identified by disruption; it possesses a leucine zipper motif.

Replication origins and pause sites in sea urchin mitochondrial DNA.

We have used a combination of one- and two-dimensional agarose gel electrophoresis, and solution hybridization to strand-specific probes, to map the replication origin of sea urchin mitochondrial DNA and to investigate the structure of replication intermediates. These assays are consistent with replication initiating unidirectionally from the D-loop region by D-loop expansion, as in vertebrates. A prominent site of initiation of lagging-strand synthesis lies at, or near to, the boundary between the genes for ATPase 6 and COIII, which is also close to a pause site for leading-strand synthesis. These findings suggest a role for pause sites in the regulation of mitochondrial transcription and replication, possibly involving template-binding proteins.

DNA binding properties of an HMG1-related protein from yeast mitochondria.

The DNA binding properties of ABF2, an abundant protein found in the mitochondria of the yeast Saccharomyces cerevisiae have been examined in detail. ABF2 is closely related to the vertebrate high mobility group protein HMG1 and like HMG1, ABF2 will introduce negative supercoils into a relaxed, double-stranded circular DNA molecule in cooperation with a DNA topoisomerase. Additionally, ABF2 binds approximately 5-10 times more tightly to negatively supercoiled DNA than to relaxed circular or linear DNA. Although ABF2 binds to most random double-stranded sequences with roughly equal affinity, its binding within certain key regulatory regions is qualitatively quite different. First, ABF2 binding induces a distinct pattern of DNA bending within the chromosomal origin of DNA replication, ARS1. Second, ABF2 binding to all nuclear replication origins tested, in addition to a critical mitochondrial promoter and replication origin, is clearly nonrandom as visualized by DNase1 footprinting. Analysis of the sequences found within these regions as well as competition experiments with synthetic DNA molecules suggest that site-specific DNA binding may be accomplished by the phased distribution of short stretches of poly(dA), which exclude ABF2 binding. These patterns of ABF2 DNA binding suggest a role for the protein in genome organization and site-specific regulation of transcription or DNA replication.

Mitochondrial DNA replication and transcription are dissociated during embryonic cardiac hypertrophy.

Cardiac hypertrophy was produced in embryonic chicks by decreasing the incubation temperature from 38 degrees C to 32 degrees C on day 11. Increases in ventricular protein, RNA, and DNA support the cardiac enlargement. Cytochrome-c oxidase activity and citrate synthase activity were depressed in hypothermic ventricles by 63% and 56%, respectively. No significant differences were seen in enzyme activities in pectoralis muscles. The involvement of mitochondrial gene replication and transcription was evaluated using a cDNA clone for the mitochondrially encoded subunit III of cytochrome-c oxidase (CO III). Quantitative slot-blot analysis demonstrated that the relative CO III mRNA concentration was reduced in hypothermic ventricles. In contrast, the relative mitochondrial DNA concentration was increased in hypothermic ventricles. Taken together, these data indicate that a hypothermia-induced decrease in cytochrome-c oxidase activity is associated with a decrease in CO III mRNA, which is not coupled to a decrease in the mitochondrial DNA copy number. This dissociation of mitochondrial gene replication and transcription may provide a useful model for examining the regulation of mitochondrial biogenesis.

Peripheral benzodiazepine induces morphological changes and proliferation of mitochondria in glioma cells.

Peripheral benzodiazepine (PBD) receptors are localized on the mitochondrial membrane and are highly expressed in brain tumors compared to normal brain. To elucidate the biological role of the PBD receptor on mitochondria, we examined the effect of PBDs on mitochondrial morphology in C6 and T98G glioma cells using rhodamine 123 and quantitative electron microscopy. In cells incubated in serum-free medium alone, mitochondria were distributed in a filamentous pattern throughout the cytoplasm. By contrast, the mitochondria aggregated in the perinuclear region in PK11195 or Ro5-4864 (10 nM) treated cells. Quantitative electron micrography revealed a 250% increased in the number of mitochondria with elongated cristae and a fivefold increase in dividing mitochondria in PK11195-treated cells compared with cells incubated in serum-free medium alone. PBD treatment also resulted in vacuolation within the matrix and mitochondrial swelling. These data suggest that PBDs influence mitochondrial morphology and induce mitochondrial replication in cultured glioma cells.

Identification of two homologous mitochondrial DNA sequences, which bind strongly and specifically to a mitrochondrial protein ofParacentrotus lividus

Using a combination of band shift and DNasel protection experiments, two Paracentrotus lividus mitochondrial sequences, able to bind tightly and selectively to a mitochondrial protein from sea urchin embryos, have been found. The two sequences, which compete with each other for binding to the protein, are located in two genome regions which are thought to contain regulatory signals for mitochondrial replication and transcription. A computer analysis suggests that the sequence TTTTRTANNTCYYATCAYA, common to the two binding regions, is the minimal recognition signal for the binding to the protein. We discuss the hypothesis that the protein binding capacity of these two sequences is involved in the control of sea urchin mtDNA replication during developmental stages.

Effects of hap mutations on heme and cytochrome formation in yeast

Simultaneous effects of mutations in the transcriptional regulatory genes, HAP1, HAP2 and HAP3, on all respiratory cytochromes of Saccharomyces cerevisiae were determined. Cytochrome behavior in hap mutants and in cyc4 and rhm1 mutants, altered in regulation of 5-aminolevulinate synthase, was compared. Although hap mutants were isolated as trans-acting, transcriptional regulators of the CYC1 (iso-1-cytochrome c) gene, each mutant exhibits partial deficiencies in all cytochrome types. In hap2 and hap3 strains all cytochromes were decreased proportionally to about 40-50% of wild type values. In contrast, hap1 caused a decrease in all cytochromes and an accumulation of a pigment, probably Zn porphyrin. Apparently apocytochrome and heme biosynthesis retain coordination in hap2 and hap3, but not in hap1, mutants. Unlike cyc4 and rhm1 mutants, hap mutants do not exhibit 5-aminolevulinate-dependent restoration of cytochromes. The hap1 mutant grew at near-normal rates on glycerol, whereas hap2 and hap3 mutants grew very slowly. The frequency of [rho-] was high (16-18%) in hap2 and hap3 strains. Results are consistent with generalized control of mitochondrial replication directed by the HAP1-HAP2 system and heme-directed control of formation of all apocytochromes mediated by HAP1. Neither system exerts all-or-nothing control.

Interaction of DNA polymerases with phospholipids

Effects of various phospholipids on the in vitro reactions of eukaryotic DNA polymerases α, β and γ were tested systematically. When phospholipids were added directly to the direction mixture, neither stimulation nor inhibition was produced. However, when phospholipids were preincubated with enzymes in the absence of template-primer, some of them showed strong inhibition. Cardiolipin strongly inhibited the reactions of all three DNA polymerases and also of terminal deoxynucleotidyl transferase. Phosphatidylinositol selectively inhibited the reaction of DNA polymerase γ. Phosphatidic acid moderately inhibited DNA polymerase α and strongly inhibited DNA polymerase γ. The inhibition of DNA polymerase γ by cardiolipin was nearly competitive with template-primer. Since the inhibition was reversed by the addition of 0.05% Triton-X 100 during preincubation, the phospholipid might interact with enzyme protein at the hydrophobic region in competition with template-primer. These results suggest a possible involvement of phospholipids in DNA replication in mitochondria and in nucleus through interaction with DNA polymerase.

DNA curvature in front of the human mitochondrial L-strand replication origin with specific protein binding

DNA bending has been suggested to play a role in the regulation of gene expression, initiation of DNA-replication, site specific recombination, and DNA packaging. In the human mitochondrial DNA we have found a DNA curvature structure within the 3'-region of ther URF2 sequence in front of the L-strand origin of replication. This structure interacts specifically with a protein factor isolated from mitochondria. Based on the localization of this DNA curvature structure and the known function of such structures the data suggest a model in which this DNA signal sequence and its specific protein binding is involved in the regulatory initiation event of L-strand replication.

A nuclear gene essential for mitochondrial replication suppresses a defect of mitochondrial transcription in Saccharomyces cerevisiae.

A genomic DNA fragment from yeast was isolated by transforming a temperature sensitive pet mutant. This mutant, pet-ts 798, has previously been characterized by its altered mitochondrial transcription apparatus. Subcloning and DNA sequencing of the genomic DNA fragment identified a reading frame responsible for the restoration of the pet-ts phenotype. The reading frame of 1023 bp is transcribed as an RNA of about 1100 nucleotides. The putative protein of 40 kDa possesses a hydrophobic amino-terminus and acidic and basic domains characteristic of recently described transcriptional activators. The inactivation of the functional gene by the introduction of an insertion fragment into the reading frame, leads to a stable pet phenotype. Further analysis of this mutant created by gene disruption makes clear that the respiratory defect is caused by the complete loss of mitochondrial DNA. Experimental evidence is given that the cloned gene acts as an intergenic suppressor of the mutant pet-ts 798. Therefore, the isolated gene represents a new factor involved in the regulation of mitochondrial replication and transcription.

Applications of the twist difference to DNA structural analysis.

The twist is a fundamental geometric property of nucleic acids. Calculation of the twist in the most general case requires detailed specification of the three-dimensional path of each strand, but many important cases may be analyzed by considering only the twist difference. If C1, C2, and C3 are three distinct space curves, the twist difference about C1 is defined as Tw(C3, C1) - Tw(C2, C1). We show here that this difference measures the rotation of the correspondence surface joining C1 to C2 about the correspondence surface joining C1 to C3. This result has application to DNA containing local nonuniformities, such as denatured regions, cruciforms, and other altered structures. It also facilitates the calculation of twist for three-stranded structures, including D-loops in mitochondrial DNA and replication and transcription intermediates. The twist difference may also be used to simplify greatly the analysis of twist changes in duplex DNA due to winding on surfaces, such as histones and certain enzymes. In such cases the strand-axis twist of DNA divides into two independent terms. The first term arises from the twist of the local reference frame, and the second arises from the rotation of either strand about the duplex axis as measured in the local reference frame. Twist changes consequent to nucleosome winding, for example, arise from the twist of the nucleosome axis, a straight line, about the DNA axis plus the rotation of either strand of the DNA about its axis in the reference frame of the cylinder.

Specificity factor of yeast mitochondrial RNA polymerase. Purification and interaction with core RNA polymerase.

We have identified a mitochondrial protein from Saccharomyces cerevisiae which confers the ability to recognize mitochondrial promoters onto a nonspecifically transcribing mitochondrial core RNA polymerase and we have purified this specificity factor 10,700-fold from a whole cell extract. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fraction followed by elution and renaturation of protein activity shows that the specificity factor is a 43-kDa polypeptide which directs mitochondrial core RNA polymerase to promoters belonging to rRNA-, tRNA-, and protein-encoding genes, as well as to mitochondrial replication origins. Gel filtration and glycerol gradient sedimentation studies indicate that the specificity factor shows little association with core RNA polymerase in the absence of DNA, and that it behaves like a monomeric 43-kDa protein.

The inhibition of mitochondrial DNA replication in vitro by the metabolites of benzene, hydroquinone and p-benzoquinone

Rat liver mitochondria incubated with the metabolites of benzene, p-benzoquinone or 1,2,4-benzenetriol, showed a dose-dependent inhibition of [3H]dTTP incorporation into mtDNA with median inhibitory concentrations of 1 mM for each compound. Benzene and the metabolites phenol, catechol and hydroquinone did not inhibit at concentrations up to 10 mM. Similarly, incubation of p-benzoquinone or hydroquinone with rabbit bone marrow mitochondria showed a dose-dependent inhibition of mtDNA synthesis with 50% inhibition at 1 mM and 10 mM, respectively. That these metabolites inhibit mitochondrial replication was evidenced by the fact that [3H]dTTP incorporation into characteristic 38S, 27S and 7S mitochondrial replication intermediates was decreased by the quinones, as analyzed on 5-20% neutral sucrose velocity gradients. p-Benzoquinone, hydroquinone and 1,2,4-benzenetriol inhibited the activity of partially purified rat liver mtDNA polymerase gamma using either activated calf thymus DNA or poly(rA) X p(dT)12-18 as primer/template, with 50% inhibitory concentrations of 25 microM, 25 microM and 180 microM, respectively. Preincubation of the metabolites with polymerase gamma or primer/template, followed by removal of the unreacted metabolite by gel filtration, indicated that inhibition resulted from interaction of the metabolites with the enzyme, rather than with the template. Binding appeared to involve a sulfhydryl residue on the enzyme since the binding of [14C]hydroquinone was prevented by N-ethylmaleimide. The ability of hydroquinone or p-benzoquinone to inhibit binding of [14C]hydroquinone to the enzyme suggests that the compounds bind to a common site or are converted to a common intermediate. Inhibition of, or changes in, replication in mitochondria of bone marrow cells by hydroquinone and p-benzoquinone may explain the changes in the mitochondrial genome observed in marrow stem cells in acute myelogenous leukemia and may suggest a mechanism for benzene leukemogenesis.

Cloning, physical mapping and genome organization of mitochondrial DNA from Cyprinus carpio oocytes.

The mitochondrial genome from Cyprinus carpio oocytes is a 10.5 megadalton, circular DNA molecule. The carp mitochondrial DNA was cloned in pBR325. Three recombinant plasmids accounted for the entire genome. Mapping of this DNA using 11 different restriction endonucleases is reported here. Both the large and small rRNA genes were then localized using Southern blot analysis. The subunit I of the cytochrome oxidase, the cytochrome b, the tRNAGlu and the URF 4 genes were localized by nucleotide sequence analysis and homology studies with human mtDNA. Our results suggest that a similar gene order has been maintained in the mitochondrial genomes of Chordata and support the hypothesis of a common ancestor for all vertebrate organelle genomes. This study constitutes the first report on the genome organization of a fish mtDNA and provides information for further investigation in connection with sequence determination, replication, and gene expression in carp mitochondria.

The orir to ori+ mutation in spontaneous yeast petites is accompanied by a drastic change in mitochondrial genome replication

The ori r petite mutants of Saccharomyces cerevisiae show a very low level of suppressivity (5–12%; suppressivity is the percentage of diploid petites issued from a cross of the parental haploid petite with a wild-type cell), indicating a poor replication efficiency of their mitochondrial genome. The latter is made up of repeat units containing two inverted ori sequences and arranged as tandem pairs in inverted orientation relative to their nearest neighbors. After subcloning ori r petites or crossing with wild-type cells a large number of ori + petites are found in the progeny. In contrast to the ori r petites, from which they are derived, these ori + petites are characterized by high suppressivity levels (approx. 90%) and contain mitochondrial genomes made up of tandem repeat units containing single ori sequences. The structural changes underlying the ori r to ori + mutation are therefore accompanied by a dramatic increase in suppressivity, indicating that the elimination of inverted ori sequences causes a drastic change from very poor to very good replicative efficiency in the mitochondrial genome. Finally, crosses of ori o petites with wild-type cells were also studied; the results obtained have clarified the reasons for the high frequency of petites having genomes similar to those of ori r petites after mutagenesis with ethidium bromide.

Selective loss of mitochondrial genome can be caused by certain unsaturated fatty acids

Various unsaturated fatty acids had different effectiveness for maintaining the continued replication of functional mitochondria in an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae (KD115). Certain isomers of octadecenoic acid (i.e., cis-9) and eicosatrienoic acid (i.e., cis-8,11,14) permitted continued replication of mitochondria and provided cultures that contained only 4 to 5% cells that formed petite colonies. On the other hand, cultures grown with cis-12- or cis-13-octadecenoic acid or cis-11,14,17-eicosatrienoic acid, produced a 12- to 16-fold greater frequency of petite mutants (50–60%) after 8 to 10 generations of growth. The production of the petite mutants occurred despite adequate incorporation of these unsaturated fatty acids into cellular phospholipids and an apparently normal ability to undergo the initial steps in the induction of cellular respiration. The evidence suggests that some cellular processes necessary for continued mitochondrial replication depend on the structural features of the fatty acyl chains as well as the overall content of unsaturated fatty acids in membrane phospholipids. Impairment of that process by certain inadequate fatty acids or by an inadequate supply of a suitable fatty acid leads to a permanent loss of the mitochondrial genome from the cells of subsequent generations.

The nucleus negatively controls the synthesis of mitochondrial proteins in the sea urchin egg

Enucleation of Paracentrotus lividus eggs, followed by partheno-genetic activation induces a sharp increase in the synthesis of mitochondrial proteins as shown by electrofluorography after in vivo labeling with radioactive amino acids. These results further substantiate the hypothesis that the cell nucleus negatively controls mitochondrial replication in the sea urchin egg.

DNA sequence of the Xenopus laevis mitochondrial heavy and light strand replication origins and flanking tRNA genes.

We have determined the primary structure of the two regions of the Xenopus laevis mitochondrial genome which encompass the origins of heavy (H) and light (L) strand replication. The first segment, which consists of 2398 nucleotides, contains the displacement loop (D-loop), the tRNA genes for threonine, proline and phenylalanine, the origin of H-strand replication, and the promoters of H- and L-strand transcription. The second segment, which consists of 447 nucleotides, contains the L-strand replication origin flanked by the tRNA genes for tryptophan, alanine, asparagine, cysteine, and tyrosine. A comparison of the sequences of the Xenopus laevis mitochondrial L-strand replication origin region and the eight tRNA genes with their counterparts from the mammalian mitochondrial genomes reveals that these regions are quite homologous, while its D-loop region shows only slight homology with those of the mammalian mitochondrial genomes.