Pulse-label analysis and mapping of the two terminal regions of asynchronous complementary strand replication of mitochondrial DNA in transformed hamster cells.
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Animals↗
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Results for “Mitochondrial DNA replication”
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The putative origin of heavy strand replication (oriH) in mitochondrial DNA is highly conserved among the teleost fishes.
We have determined the nucleotide sequence of a 645 bp EcoRI-HindIII fragment from mitochondrial DNA of Arctic charr, Salvelinus alpinus. The sequence includes the tRNA(Phe) gene, part of the small subunit ribosomal RNA gene as well as the putative origin of heavy strand replication located at the major non-coding region, the D-loop containing region. Upon comparison of the Arctic charr sequence to mitochondrial DNA sequences from several distantly related teleost fishes, the origin of heavy strand replication was found to be highly conserved among the teleost fishes.
Animals↗
Response characteristics of the mitochondrial DNA genome in developmental health and disease.
This review focuses on mitochondrial biology in mammalian development; specifically, the dynamics of information transfer from nucleus to mitochondrion in the regulation of mitochondrial DNA genomic expression, and the reverse signaling of mitochondrion to nucleus as an adaptive response to the environment. Data from recent studies suggest that the capacity of embryonic cells to react to oxygenation involves a tradeoff between factors that influence prenatal growth/development and postnatal growth/function. For example, mitochondrial DNA replication and metabolic set points in nematodes may be determined by mitochondrial activity early in life. The mitochondrial drug PK11195, a ligand of the peripheral benzodiazepine receptor, has antiteratogenic and antidisease action in several developmental contexts in mice. Protein malnutrition during early life in rats can program mitochondrial DNA levels in adult tissues and, in humans, epidemiological data suggest an association between impaired fetal growth and insulin resistance. Taken together, these findings raise the provocative hypothesis that environmental programming of mitochondrial status during early life may be linked with diseases that manifest during adulthood. Genetic defects that affect mitochondrial function may involve the mitochondrial DNA genome directly (maternal inheritance) or indirectly (Mendelian inheritance) through nuclear-coded mitochondrial proteins. In a growing number of cases, the depletion of, or deletion in, mitochondrial DNA is seen to be secondary to mutation of key nuclear-coded mitochondrial proteins that affect mitochondrial DNA replication, expression, or stability. These defects of intergenomic regulation may disrupt the normal cross-talk or structural compartmentation of signals that ultimately regulate mitochondrial DNA integrity and copy number, leading to depletion of mitochondrial DNA.
Animals↗
Properties of a primer RNA-DNA hybrid at the mouse mitochondrial DNA leading-strand origin of replication.
Primers for vertebrate mitochondrial leading-strand DNA replication are products of transcription synthesized by mitochondrial RNA polymerase. The precursor primer RNA exists as a persistent RNA-DNA hybrid, known as an R-loop, formed during transcription through the replication origin (Xu, B., and Clayton, D. A. (1996) EMBO J. 15, 3135-3143). In an effort to examine the precise structure of this primer RNA intermediate, we have used two methods to reconstitute model R-loops containing the mouse mitochondrial DNA origin sequence. First, we demonstrate that bacteriophage SP6 RNA polymerase can efficiently catalyze the formation of an R-loop at the mouse mtDNA origin sequence. Second, the R-loop can be assembled by annealing presynthesized RNA and supercoiled DNA template in the presence of formamide. R-loop formation by either method is dependent on specific template sequences. The reconstituted R-loop is exceptionally stable and exhibits an unexpected structure. Structural studies indicate that the RNA strand is organized within the RNA-DNA base-paired region, suggesting that the heteroduplex interaction occurs through a specific conformation. We propose that the organized structure of the R-loop is critical for primer RNA function in vivo with important implications for the RNA processing and DNA replication machinery.
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Saccharomyces cerevisiae contains an RNase MRP that cleaves at a conserved mitochondrial RNA sequence implicated in replication priming.
Yeast mitochondrial DNA contains multiple promoters that sponsor different levels of transcription. Several promoters are individually located immediately adjacent to presumed origins of replication and have been suggested to play a role in priming of DNA replication. Although yeast mitochondrial DNA replication origins have not been extensively characterized at the primary sequence level, a common feature of these putative origins is the occurrence of a short guanosine-rich region in the priming strand downstream of the transcriptional start site. This situation is reminiscent of vertebrate mitochondrial DNA origins and raises the possibility of common features of origin function. In the case of human and mouse cells, there exists an RNA processing activity with the capacity to cleave at a guanosine-rich mitochondrial RNA sequence at an origin; we therefore sought the existence of a yeast endoribonuclease that had such a specificity. Whole cell and mitochondrial extracts of Saccharomyces cerevisiae contain an RNase that cleaves yeast mitochondrial RNA in a site-specific manner similar to that of the human and mouse RNA processing activity RNase MRP. The exact location of cleavage within yeast mitochondrial RNA corresponds to a mapped site of transition from RNA to DNA synthesis. The yeast activity also cleaved mammalian mitochondrial RNA in a fashion similar to that of the mammalian RNase MRPs. The yeast endonuclease is a ribonucleoprotein, as judged by its sensitivity to nucleases and proteinase, and it was present in yeast strains lacking mitochondrial DNA, which demonstrated that all components required for in vitro cleavage are encoded by nuclear genes. We conclude that this RNase is the yeast RNase MRP.
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Origin and direction of replication in mitochondrial DNA molecules from the genus Drosophila.
Mitochondrial DNA (mtDNA) obtained from ovaries of Drosophila simulans, D. mauritiana, D. takahashii, D. yakuba and D. virilis was examined by electron microscopy. From a consideration of the structural properties of replicative intermediates, it was concluded that in mtDNA molecules of each species, synthesis on one strand can be up to 97% complete before synthesis on the complementary strand is initiated. MtDNA molecules of each species contain a single A+T-rich region which shows species-specific size variation from 1.0 kb (D. virilis) to 4.8 kb (D. simulans), and maps at the same position in all molecules relative to three common EcoRI sites. The structural properties of complex forms, interpreted as having originated from replicative intermediates, and produced by either partial denaturation or EcoRI digestion, are consistent with the hypothesis that replication is initiated within the A+T-rich region and proceeds unidirectionally around the molecule towards the nearest common EcoRI site. The replication origin is located near the center of the A+T-rich region in D. simulans and D. mauritiana, but lies closer to that end of the A+T-rich region which is distal to the nearest common EcoRI site in D. takahashii, D. yakuba and D. virilis.
Animals↗
Mode of extension of the daughter strands in the replication of closed circular mitochondrial DNA in vitro.
1. The properties of nascent DNA in the replicative closed circular mitochondrial DNA were examined in the in vitro system of discontinuous replication, using newborn rat liver mitochondria containing endogeneous DNA templates and enzymes. 2. The nascent DNA associated with the closed circular DNA fraction was found to be of two types; one class consisted of the fragments, and the other of the higher molecular-weight DNAs. Data from pulse and chase experiments indicate that the fragments were initially synthesized and subsequently converted into both heavy and light strands of the higher molecular-weight DNAs in an asymmetrical mode. 3. DNA - DNA hybridization experiments revealed that half of the fragments at least were copies of complementary parts of the parental DNA. 4. Based on the present in vitro data, a tentative structure of the replicating region and its expansion are discussed.
Animals↗
Replication and transcription of mammalian mitochondrial DNA.
Mitochondria are subcellular organelles, devoted mainly to energy production in the form of ATP, that contain their own genetic system. Mitochondrial DNA codifies a small, but essential number of polypeptides of the oxidative phosphorylation system. The mammalian mitochondrial genome is an example of extreme economy showing a compact gene organization. The coding sequences for two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and 13 polypeptides are contiguous and without introns. The tRNAs are regularly interspersed between the rRNA and protein-coding genes, playing a crucial role in RNA maturation from the polycistronic transcripts. A single major non-coding region, called the D-loop region, contains the main regulatory sequences for transcription and replication initiation. This genetic organization has its precise correspondence in the mode of expression and distinctive structural features of the RNAs. The basic mechanisms of mitochondrial DNA transcription and replication and the main cis-acting elements playing a role in both processes have been determined. Many trans-acting factors involved in mitochondrial gene expression, including the RNA and DNA polymerases, have been cloned or identified. However, the regulatory mechanisms participating in mitochondrial gene expression are still poorly understood. The interest to complete this knowledge is increased by the involvement of mitochondria in human diseases, in basic processes such as heat production, Ca(2+) homeostasis and apoptosis, and by their potential role in ageing and carcinogenesis.
Animals↗
DNA-helicase activity from sea urchin mitochondria.
As a step toward the characterization of the main components of mitochondrial DNA replication apparatus in sea urchin, we report the identification of a DNA-helicase activity in Paracentrotus lividus mitochondria. The activity was detected in a protein fraction obtained by fractionating on DEAE-Sephacel a lysate of gradient purified mitochondria from paracentrotus lividus eggs. The mitochondrial helicase unwound, in the presence of ATP and Mg++, a 39-base oligonucleotide annealed to single-stranded M13mp18 (+) DNA. Its direction of movement is 3' to 5' with respect to the single stranded portion of the partial duplex DNA substrate. This polarity is similar to that exhibited by the Escherichia coli rep helicase and by the helicase from bovine brain mitochondria. These features suggest that the sea urchin mitochondrial helicase could function in enabling the polymerization of the H-strand during mitochondrial DNA replication.
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Reconstitution of a minimal mtDNA replisome in vitro.
We here reconstitute a minimal mammalian mitochondrial DNA (mtDNA) replisome in vitro. The mtDNA polymerase (POLgamma) cannot use double-stranded DNA (dsDNA) as template for DNA synthesis. Similarly, the TWINKLE DNA helicase is unable to unwind longer stretches of dsDNA. In combination, POLgamma and TWINKLE form a processive replication machinery, which can use dsDNA as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. The addition of the mitochondrial ssDNA-binding protein stimulates the reaction further, generating DNA products of about 16 kb, the size of the mammalian mtDNA molecule. The observed DNA synthesis rate is 180 base pairs (bp)/min, corresponding closely to the previously calculated value of 270 bp/min for in vivo DNA replication. Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. Furthermore, mutations in TWINKLE and POLgamma cause autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with deletions in mitochondrial DNA. The functional interactions between TWINKLE and POLgamma thus explain why mutations in these two proteins cause an identical syndrome.
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Structural and replicative forms of mitochondrial DNA from human leukocytes in relation to age.
The structure and replication of human leukocyte mitochondrial DNA (mtDNA) was investigated in healthy young adult males (23--37 years old), middle-aged males (42--52 years old) with secondary polycythemia, and elderly males (80--89 years old) who exhibited different degrees of age-related disease syndromes. The distribution of the various cell types within the white cell population was within normal limits in all samples. Total mtDNA was isolated in ethidium bromide--CsCl gradients and examined by electron microscopy after spreading by the aqueous and formamide techniques. The individual frequencies of catenated forms ranged from 2 to 6% but showed relatively little change (declining slightly) with age. The individual frequencies of circular dimers varied from 0 to 0.1% in the young adult and polycythemic groups and in 10 out of 12 elderly individuals. One elderly individual had a circular dimer frequency of 0.3% (including a circular molecular of tetramer size) and another had 4.5%. This finding suggest that agerelated cellular pathology may exist in the blood-forming system in some cases. The mode of replication of leukocyte mtDNA agrees well with that described for mouse L cells. There was no evidence of aberrant mtDNA replication as a result of aging.
Adult↗
Replication of animal mitochondrial DNA.
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Animals↗
A deoxyribonucleotidase in mitochondria: involvement in regulation of dNTP pools and possible link to genetic disease.
Three cytosolic and one plasma membrane-bound 5'-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5'-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5'- and 2'(3')-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith-Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease.
5'-Nucleotidase↗
DNA synthesis in a mitochondrial lysate of Xenopus laevis oocytes. H strand replication in vitro.
Conditions for efficient replication in vitro of mitochondrial DNA L strand into H strand products have been established. Gel electrophoresis and hybridization analyses of the products show that neosynthesized H strands are progressively elongated from the D-loop region, and some of them are synthesized as full-length molecules. Evidence for initiation of these H strands de novo is presented. In contrast, there is no detectable L strand synthesis in vitro in this system. This may prove useful for analyzing the distinct molecular mechanisms operating at OH and OL. Use of specific inhibitors indicates that DNA synthesis in the mitochondrial lysate in vitro requires DNA polymerase gamma. These observations support the conclusion that replication in vitro in this system closely resembles the first steps of mitochondrial DNA replication in vivo.
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Intermediates in the replication of the mitochondrial DNA of Tetrahymena pyriformis.
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Animals↗
Release of replication termination controls mitochondrial DNA copy number after depletion with 2',3'-dideoxycytidine.
Although cellular mitochondrial DNA (mtDNA) copy number varies widely among cell lines and tissues, little is known about the mechanism of mtDNA copy number control. Most nascent replication strands from the leading, heavy-strand origin (O(H)) are prematurely terminated, defining the 3' boundary of the displacement loop (D-loop). We have depleted mouse LA9 cell mtDNA to approximately 20% of normal levels by treating with 2',3'-dideoxycytidine (ddC) and subsequently allowed recovery to normal levels of mtDNA. A quantitative ligation-mediated PCR assay was used to determine the levels of both terminated and extended nascent O(H) strands during mtDNA depletion and repopulation. Depleting mtDNA leads to a release of replication termination until mtDNA copy number approaches a normal level. Detectable total nascent strands per mtDNA genome remain below normal. Therefore, it is likely that the level of replication termination plays a significant role in copy number regulation in this system. However, termination of D-loop strand synthesis is persistent, indicating formation of the D-loop structure has a purpose that is required under conditions of rapid recovery of depleted mtDNA.
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Polγ coordinates DNA synthesis and proofreading to ensure mitochondrial genome integrity.
Accurate replication of mitochondrial DNA (mtDNA) by DNA polymerase γ (Polγ) is essential for maintaining cellular energy supplies, metabolism, and cell cycle control. To illustrate the structural mechanism for Polγ coordinating polymerase (pol) and exonuclease (exo) activities to ensure rapid and accurate DNA synthesis, we determined four cryo-EM structures of Polγ captured after accurate or erroneous incorporation to a resolution of 2.4-3.0 Å. The structures show that Polγ employs a dual-checkpoint mechanism to sense nucleotide misincorporation and initiate proofreading. The transition from replication to error editing is accompanied by increased dynamics in both DNA and enzyme, in which the polymerase relaxes its processivity and the primer-template DNA unwinds, rotates, and backtracks to shuttle the mismatch-containing primer terminus 32 Å to the exo site for editing. Our structural and functional studies also provide a foundation for analyses of Polγ mutation-induced human diseases and aging.
Humans↗
Photoaging-associated large-scale deletions of mitochondrial DNA.
Heteroplasmy, replicative segregation, low copy numbers of mtDNA, and selection mechanisms at the molecular and cellular level are all factors that determine requirements toward the experimental design for the detection and the quantification of mtDNA mutations. The short half-life and low stability of ROS further increase the technical demands. However, the continuous improvement of techniques has given us more insight into the interactions between ROS and mtDNA, both at the level of endogenous ROS produced by the normal mitochondrial metabolism and exogenous sources of ROS, such as singlet oxygen, which can result from treatments such as UVA exposure.
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