Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Mitochondrial DNA replication”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14Linked to original sources
PubMed2002

Biased incorporation of ribonucleotides on the mitochondrial L-strand accounts for apparent strand-asymmetric DNA replication.

Recently, we presented evidence for conventional, strand-coupled replication of mammalian mitochondrial DNA. Partially single-stranded replication intermediates detected in the same DNA preparations were assumed to derive from the previously described, strand-asymmetric mode of mitochondrial DNA replication. Here, we show that bona fide replication intermediates from highly purified mitochondria are essentially duplex throughout their length, but contain widespread regions of RNA:DNA hybrid, as a result of the incorporation of ribonucleotides on the light strand which are subsequently converted to DNA. Ribonucleotide-rich regions can be degraded to generate partially single-stranded molecules by RNase H treatment in vitro or during DNA extraction from crude mitochondria. Mammalian mitochondrial DNA replication thus proceeds mainly, or exclusively, by a strand-coupled mechanism.

Animals↗
PubMed2003

Failure to produce mitochondrial DNA results in embryonic lethality in Rnaseh1 null mice.

Although ribonucleases H (RNases H) have long been implicated in DNA metabolism, they are not required for viability in prokaryotes or unicellular eukaryotes. We generated Rnaseh1(-/-) mice to investigate the role of RNase H1 in mammals and observed developmental arrest at E8.5 in null embryos. A fraction of the mainly nuclear RNase H1 was targeted to mitochondria, and its absence in embryos resulted in a significant decrease in mitochondrial DNA content, leading to apoptotic cell death. This report links RNase H1 to generation of mitochondrial DNA, providing direct support for the strand-coupled mechanism of mitochondrial DNA replication. These findings also have important implications for therapy of mitochondrial dysfunctions and drug development for the structurally related RNase H of HIV.

Alleles↗
PubMed1996

Recombination associated with replication of malarial mitochondrial DNA.

Mitochondrial DNA of the malarial parasite Plasmodium falciparum comprises approximately 20 copies per cell of a 6 kb genome, arranged mainly as polydisperse linear concatemers. In synchronous blood cultures, initiation of mtDNA replication coincides with the start of the 4-5 doublings in nuclear DNA that mark the reproductive phase of the erythrocytic cycle. We show that mtDNA replication coincides with a recombination process reminiscent of the replication mechanism used by certain bacteriophages and plasmids. The few circular forms of mtDNA which are also present do not replicate by a theta mechanism, but are themselves the product of recombination, and we propose they undergo rolling circle activity to generate the linear concatemers.

Animals↗
PubMed1997

Mitochondrial electron transport chain activity, but not ATP synthesis, is required for drug-induced apoptosis in human leukaemic cells: a possible novel mechanism of regulating drug resistance.

There is increasing evidence for an association between mitochondrial function and susceptibility to apoptosis. It has been shown that the vinblastine-resistant leukaemic cell line CEM/VLB100 has a more active mitochondrial electron transport chain (ETC) than the parental CCRF-CEM cell line. Inhibition of mitochondrial DNA replication by ethidium bromide (EB) depleted the activity of the ETC and reduced cellular respiratory rate. Depletion of mitochondrial DNA was associated with increased resistance to vinblastine-induced apoptosis in both cell lines. In contrast, the highly specific inhibitor of the energy producing mitochondrial enzyme F1Fzero-ATPase, oligomycin, rendered CEM/VLB100 cells more sensitive to vinblastine by inhibiting the energy-dependent P-glycoprotein (Pgp) pump, suggesting that the effect of EB is independent of energy generation and ATPase activity. Both mitochondrial ETC depletion and ATPase inhibition decreased vinblastine-induced cell cycle changes in the CCRF-CEM cell line, suggesting that cell cycle changes are dependent on ATP generation. However, EB-induced ETC depletion in CEM/VLB100 cells inhibited apoptosis in response to high concentration of vinblastine, but not G2M arrest. We suggest that: (1) over-expression of Pgp by drug-resistant cells may up-regulate mitochondrial energy production; (2) mitochondrial ETC activity is required for DNA fragmentation in response to vinblastine, but the mechanism is independent of Pgp activity and ATP generation; (3) down-regulation of mitochondrial ETC activity may confer resistance to vinblastine-induced apoptosis; (4) the mitochondrial ETC is involved in vinblastine-induced apoptosis downstream of microtubule disruption and cell cycle changes.

Adenosine Triphosphatases↗
PubMed1988

Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo.

A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4 degrees C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases. These studies show that chromosomal DNA replication in isolated nuclei is mediated by stable replication forks that are assembled in a temporally specific fashion in vivo and indicate that the synthetic mechanisms observed in vitro accurately reflect those operative in vivo.

Animals↗
PubMed1976

Biogenesis of mitochondria. XLII. Genetic analysis of the control of cellular mitochondrial DNA levels in Saccharomyces cerevisiae.

The proportion of total cell DNA which is mitochondrial DNA was measured in haploid, diploid and tetraploid strains of S. cerevisiae grown under a standard set of conditions. For all strains tested the mitochondrial DNA level was in the range 16%-25% of total cell DNA. Repeated measurements of the cellular level of mitochondrial DNA in two haploid strains showed that these strains have measurably different cellular mitochondrial DNA levels (17% and 24% of total DNA, respectively) under our conditions. These two grande strains were used to investigate the role of the mitochondrial and nuclear genomes in the regulation of the mitochondrial DNA level. We have shown by genetic analysis that the difference between these two strains is determined by at least two nuclear genes. The mitochondrial genome is not involved in the regulation of cellular mitochondrial DNA levels. A number of purified petite clones derived from independent spontaneous petite isolates of the grande strain which contained 24% mitochondrial DNA were also studied. The mitochondrial DNA levels in all but one of these petites fell in the range 20-25% of total cell DNA. From these results we conclude that, in general, the mitochondrial DNA level in petite strains is controlled by the same mechanism as operates in grande strains. We propose a general model for the control of the cellular mitochondrial DNA level, in which the amount of mitochondrial DNA per cell is determined by regulation of the number of mitochondrial DNA molecules per cell. This regulation is mediated through the availability of a set of nuclear coded components, possibly a mitochondrial membrane site, which are required for the replication of mitochondrial DNA.

Aneuploidy↗
PubMed1995

RNase mitochondrial RNA processing cleaves RNA from the rat mitochondrial displacement loop at the origin of heavy-strand DNA replication.

Ribonuclease mitochondrial RNA processing cleaves RNAs from the mammalian mitochondrial main non-coding regulatory region, called the displacement loop. Our data demonstrate that rat cells contain a site-specific ribonuclease mitochondrial RNA processing activity. We found that this enzyme processes the rat mitochondrial displacement-loop RNA substrate at the level of the conserved sequence block 1, a result which is different from that for mouse. This finding correlates with the in-vivo transcriptional analysis of the rat displacement-loop region. Processing by homologous and heterologous ribonuclease mitochondrial RNA enzymes occurs in the same manner, suggesting a conserved mode of substrate recognition.

Animals↗
PubMed1996

Skeletal muscle mitochondria from AZT-treated rats have a diminished response to chronic electrical stimulation.

Inhibition of DNA polymerase gamma-function mediated by 3'-azido-3'-deoxythymidine (AZT) has been proposed to cause a myopathy by reducing mitochondrial DNA (mtDNA) replication. Repeated bouts of exercise stimulate an increase in mtDNA replication, mitochondrial content, and mitochondrial volume fraction. Therefore, adaptation of rat skeletal muscle [tibialis anterior (TA)] mitochondria exposed to AZT (1 mg/ml for 35 days) and then to electrical stimulation for 8 h/day (7, 14, 21 days) with continued AZT treatment was examined. Fourteen and 21 days of stimulation increased TA cytochrome oxidase (CO) activity, mtDNA, and CO subunit III and VIc mRNA levels in both groups. The TA CO activity and CO III mRNA increases after 14 and 21 days of stimulation were diminished in AZT-treated rats. TA glyceraldehyde-3-phosphate dehydrogenase was reduced in normal rats after chronic stimulation but was unchanged in AZT-treated rats. Chronic stimulation increased the mitochondrial volume fraction by 80 and 40% in normal and AZT-treated rats, respectively. These results indicate diminution, but not complete inhibition, of mitochondrial adaptation by AZT-treated skeletal muscle in response to stimulation.

Animals↗
PubMed2001

Mitochondrial toxicity and HIV therapy.

Nucleoside reverse transcriptase inhibitors (NRTIs) remain the cornerstone of highly active antiretroviral therapy (HAART) combination regimens. However, it has been known for some time that these agents have the potential to cause varied side effects, many of which are thought to be due to their effects on mitochondria. Mitochondria, the key energy generating organelles in the cell, are unique in having their own DNA, a double stranded circular genome of about 16 000 bases. There is a separate enzyme present inside the cell that replicates mitochondrial DNA, polymerase gamma. NRTIs can affect the function of this enzyme and this may lead to depletion of mitochondrial DNA or qualitative changes. The study of inherited mitochondrial diseases has led to further understanding of the consequences of mutations or depletion in mitochondrial DNA. Key among these is the realisation that there may be substantial heteroplasmy among mitochondria within a given cell, and among cells in a particular tissue. The unpredictable nature of mitochondrial segregation during cellular replication makes it difficult to predict the likelihood of dysfunction in a given tissue. In addition, there is a threshold effect for the expression of mitochondrial dysfunction, both at the mitochondrial and cellular level. Various clinical and in vitro studies have suggested that NRTIs are associated with mitochondrial dysfunction in different tissues, although the weight of evidence is limited in many cases. The heterogeneity in the tissues affected by the different drugs raises interesting questions, and possible explanations include differential distribution or activation of these agents. This article reviews the major recognised toxicities associated with NRTI therapy and evidence for mitochondrial dysfunction in these complications. Data were identified through searching of online databases including Medline and Current Contents for relevant articles, along with abstracts and posters from recent conferences in the HIV and mitochondrial fields.

Acidosis, Lactic↗
PubMed2002

The transmission of mitochondrial DNA following assisted reproductive techniques.

Mitochondria, among other functions, generate energy in the form of ATP. The chondrial genome, located within each mitochondrion, encodes some of the polypeptides associated with the electron transfer chain (ETC) and ATP production. Transcription and replication of mitochondrial DNA (mtDNA) is dependent upon the import of transcription and replication factors encoded by the nucleus. Certain point mutations and large-scale deletions to mtDNA can be either severely debilitating or lethal. The transmission and inheritance of mtDNA [not readable: see to offspring is strictly regulated and specific to each species. In many mammalian systems, paternal mtDNA is eliminated very early during embryonic development. However, it is possible that the paternal molecule could be extruded to those cells destined to become trophoblasts and may act as a regulator of embryonic cell fate. Furthermore, the increasing use of more sophisticated assisted reproductive techniques has led to the incorporation of extraneous mtDNA in both the reconstructed oocyte and embryo with transmission to the offspring at varying degrees.

Adenosine Triphosphate↗
PubMed1982

Unusual compartmentation of precursors for nuclear and mitochondrial DNA in mouse L cells.

Current evidence suggests that distinct mechanisms exist to regulate precursor synthesis for nuclear and mitochondrial DNA replication. We tested this is mouse L cells by asking whether nuclear and mitochondrial DNAs become labeled to equivalent specific activities when provided with an exogenous nucleic acid precursor. Cells were grown in [32P]orthophosphate-containing medium long enough to bring all pools to equivalent specific activities. [6-3H]Uridine was added to the medium as a general pyrimidine precursor. At intervals, cells were harvested and nuclear and mitochondrial DNA was isolated. After enzymatic hydrolysis of each DNA fraction to deoxyribonucleoside 5'-monophosphates, these were separated by high performance liquid chromatography and the 3H/32P ratio in each pyrimidine was determined as an index of the specific activity of DNA pyrimidine residues. The dTMP residues in nuclear and mitochondrial DNA reached roughly equal specific activities and at comparable rates. However, dCMP residues in mitochondrial DNA reached maximal specific activities more rapidly than those in nuclear DNA, and the maximal values attained were nearly twice those seen either with the nuclear DNA dCMP residues or in the dTMP residues from either DNA. This indicates that the pathways leading to dCTP synthesis are organized so that mitochondria can use exogenous precursors more effectively than can the nucleus. The nature of this compartmentation is not clear, but it evidently involves one or more steps beyond the divergence point between pathways to dCTP and dTTP.

Animals↗
PubMed1995

Mitochondrial DNA minicircles, lacking replication origins, exist in the cardiac muscle of a young normal subject.

This is the first report that mitochondrial (mt) DNA 'minicircles,' lacking replication origins, exist in the heart mtDNA of a young normal subject. A total detection system using 180 kinds of PCR primer pairs was recently devised to detect all possible deletions in mtDNA. To assess the reliability of the system, template mtDNAs were prepared from the heart muscle by three different methods including DNAase digestion. The methods for mtDNA preparation and the nuclear DNA contaminants were demonstrated to have few influences on the analysis. With not less than 95% accuracy, 52 types of deletions including 36 types of minicircles were detected in the subject's mtDNA. These results will give an insight to the mechanism of mtDNA deletion and to the relevance of the deletion with normal aging.

Adult↗
PubMed1984

Identification of autonomous replication sequences in genomic and mitochondrial DNA of Crithidia fasciculata.

High frequency transformation of Saccharomyces cerevisiae was used as a functional assay to isolate autonomous replication sequences (ars) from the genomic and kinetoplast DNA of the insect trypanosomatid Crithidia fasciculata. Three independent cloned genomic sequences and one kinetoplast DNA sequence promoted high frequency transformation and extrachromosomal maintenance of the YIp5 plasmid DNA in yeast. The kinetoplast DNA clone was sub-cloned to further localize the DNA sequence essential for ars activity. This element was shown to be contained in a 2 kb HindIII-EcoRI fragment derived from a 8 kb HindIII fragment of the maxicircle component of the kinetoplast DNA. This 2 kb fragment is within a DNA sequence that has been shown to strongly hybridize to Trypanosoma brucei maxicircle DNA.

Animals↗
PubMed1996

The cold sensitivity of a mutant of Saccharomyces cerevisiae lacking a mitochondrial heat shock protein 70 is suppressed by loss of mitochondrial DNA.

SSH1, a newly identified member of the heat shock protein (hsp70) multigene family of the budding yeast Saccharomyces cerevisiae, encodes a protein localized to the mitochondrial matrix. Deletion of the SSH1 gene results in extremely slow growth at 23 degrees C or 30 degrees C, but nearly wild-type growth at 37 degrees C. The matrix of the mitochondria contains another hsp70, Ssc1, which is essential for growth and required for translocation of proteins into mitochondria. Unlike SSC1 mutants, an SSH1 mutant showed no detectable defects in import of several proteins from the cytosol to the matrix compared to wild type. Increased expression of Ssc1 partially suppressed the cold-sensitive growth defect of the SSH1 mutant, suggesting that when present in increased amounts, Ssc1 can at least partially carry out the normal functions of Ssh1. Spontaneous suppressors of the cold-sensitive phenotype of an SSH1 null mutant were obtained at a high frequency at 23 degrees C, and were all found to be respiration deficient. 15 of 16 suppressors that were analyzed lacked mitochondrial DNA, while the 16th had reduced amounts. We suggest that Ssh1 is required for normal mitochondrial DNA replication, and that disruption of this process in ssh1 cells results in a defect in mitochondrial function at low temperatures.

Amino Acid Sequence↗
PubMed1993

RNA subunit of mitochondrial RNA-processing enzyme is induced by contractile activity in striated muscle.

A small RNA encoded within the nucleus of yeast and mammalian cells is an essential subunit of a mitochondrial RNA-processing endonuclease (RNase MRP) that generates primers for mitochondrial DNA (mtDNA) replication. We examined expression of MRP-RNA in specialized subtypes of mammalian striated muscles that differ markedly in respiratory activity and in muscles subjected to chronic stimulation via the motor nerve, a potent stimulus to mitochondrial biogenesis. MRP-RNA was more abundant in mitochondria-rich cardiac and slow-twitch skeletal muscles than in glycolytic fast-twitch skeletal muscles. Forced contractile activity resulting from nerve stimulation increased expression of MRP-RNA by 3.5-fold within the first day and by 14-fold within 14 days. Changes in abundance of MRP-RNA preceded but otherwise occurred in parallel to changes in specific activity of citrate synthase, a marker of mitochondrial proliferation shown previously to correlate with mtDNA copy number in this model. Another small RNA (U1) also was induced transiently (1-3 days) by nerve stimulation, but such changes were not sustained and were of less magnitude (< 4-fold) than changes in MRP-RNA. These findings are consistent with the hypothesis that MRP-RNA may have a regulatory function with respect to mtDNA replication and mitochondrial biogenesis.

Animals↗
PubMed1977

The action of structural analogues of ethidium bromide on the mitochondrial genome of yeast.

We have studied the effects on the yeast mitochondrial genome of four analogues of ethidium bromide, in which the phenyl moieyt has been replaced by linear alkyl chains of lengths varying from seven to fifteen carbon atoms. These analogues are more efficient than ethidium bromide in inducing petite mutants in Saccharomyces cervisiae. The drugs also cause a loss of mtDNA from the cells in vivo; however these analogues are in fact less effective inhibitors of mitochondrial DNA replication per se, as shown by direct in vitro studies. It is concluded that these analogues are more efficient than ethidium bromide in causing the fragmentation of mitochondrial DNA in S. cervisiae.

DNA, Mitochondrial↗
PubMed2006

Spatial covariation of mutation and nonsynonymous substitution rates in vertebrate mitochondrial genomes.

Mitochondrial genomes encode fundamental subunits of the basic energy producing machinery of eukaryotic cells that are under strong functional constraint. Paradoxically, these genes evolve rapidly in general, and there is substantial variation in evolutionary rates among genes within genomes. In order to investigate spatial variation in selection intensity, we conducted tests of neutrality using ratios of synonymous to nonsynonymous substitutions (dN/dS = omega) on numerous protein gene segments from fishes and mammals. Values of omega were very low for nearly all genomic regions. However, values of both omega and dN varied in a clinal pattern with increasing distance from the light-strand origin of replication. Spatial heterogeneity of nonsynonymous substitution rates exhibits a significantly positive correlation with variation in mutation rates that are related to the mode of mitochondrial DNA replication. The finding that nonsynonymous substitution rates are proportional to mutation rates is expected if a majority of substitutions are selectively neutral or slightly deleterious. Spatial patterns of among-gene variation in nonsynonymous rates were highly similar between fishes and mammals, suggesting that forces governing mitochondrial gene evolution have remained relatively constant over 450 Myr of vertebrate evolution. Conservation of substitution patterns despite major shifts in thermal habit and metabolic demands among taxa implicates a conserved replication mechanism controlling relative mutation rates as a major determinant of mitochondrial protein evolution.

Animals↗
PubMed1999

Sensitive assay for mitochondrial DNA polymerase gamma.

BACKGROUND: The mitochondrial DNA polymerase gamma is the principal polymerase required for mitochondrial DNA replication. Primary or secondary deficiencies in the activity of DNA polymerase gamma may lead to mitochondrial DNA depletion. We describe a sensitive and robust clinical assay for this enzyme. METHODS: The assay was performed on mitochondria isolated from skeletal muscle biopsies. High-molecular weight polynucleotide reaction products were captured on ion-exchange paper, examined qualitatively by autoradiography, and quantified by scintillation counting. RESULTS: Kinetic analysis of DNA polymerase gamma by this method showed a K(m) for dTTP of 1.43 micromol/L and a K(i) for azidothymidine triphosphate of 0.861 micromol/L. The assay was linear from 0.1 to 2 microgram of mitochondrial protein. The detection limit was 30 units (30 fmol dTMP incorporated in 30 min). The linear dynamic range was three orders of magnitude; 30-30 000 units. Imprecision (CV) was 6.4% within day and 12% between days. Application of this assay to a mixed population of 38 patients referred for evaluation of mitochondrial disease revealed a distribution with a range of 0-2506 U/microgram, reflecting extensive biologic variation among patients with neuromuscular disease. CONCLUSION: This assay provides a useful adjunct to current laboratory methods for the evaluation of patients with suspected mitochondrial DNA depletion syndromes.

Animals↗