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PubMed1995

Prediction and identification of new natural substrates of the yeast mitochondrial intermediate peptidase.

Most mitochondrial precursor proteins are processed to the mature form in one step by mitochondrial processing peptidase (MPP), while a subset of precursors destined for the matrix or the inner membrane are cleaved sequentially by MPP and mitochondrial intermediate peptidase (MIP). We showed previously that yeast MIP (YMIP) is required for mitochondrial function in Saccharomyces cerevisiae. To further define the role played by two-step processing in mitochondrial biogenesis, we have now characterized the natural substrates of YMIP. A total of 133 known yeast mitochondrial precursors were collected from the literature and analyzed for the presence of the motif RX(decreases)(F/L/I)XX(T/S/G)XXXX(decreases), typical of precursors cleaved by MPP and MIP. We found characteristic MIP cleavage sites in two distinct sets of proteins: respiratory components, including subunits of the electron transport chain and tricarboxylic acid cycle enzymes, and components of the mitochondrial genetic machinery, including ribosomal proteins, translation factors, and proteins required for mitochondrial DNA metabolism. Representative precursors from both sets were cleaved to predominantly mature form by mitochondrial matrix or intact mitochondria from wild-type yeast. In contrast, intermediate-size forms were accumulated upon incubation of the precursors with matrix from mip1 delta yeast or intact mitochondria from mip1ts yeast, indicating that YMIP is necessary for maturation of these proteins. Consistent with the fact that some of these substrates are essential for the maintenance of mitochondrial protein synthesis and mitochondrial DNA replication, mip1 delta yeast undergoes loss of functional mitochondrial genomes.

Amino Acid Sequence↗
PubMed2005

Replication origin of mitochondrial DNA in insects.

The precise position of the replication origin (O(R)) of mtDNA was determined for insect species belonging to four different orders (four species of Drosophila, Bombyx mori, Triborium castaneum, and Locusta migratoria, which belong to Diptera, Lepidoptera, Coleoptera, and Orthoptera, respectively). Since the free 5' ends of the DNA strands of mtDNA are interpreted as the O(R), their positions were mapped at 1-nucleotide resolution within the A + T-rich region by using the ligation-mediated PCR method. In all species examined, the free 5' ends were found within a very narrow range of several nucleotides in the A + T-rich region. For four species of Drosophila, B. mori, and T. castaneum, which belong to holometabolous insects, although the O(R)'s were located at different positions, they were located immediately downstream of a series of thymine nucleotides, the so-called T-stretch. These results strongly indicate that the T-stretch is involved in the recognition of the O(R) of mtDNA at least among holometabolous insects. For L. migratoria (hemimetabolous insect), on the other hand, none of the long stretches of T's was found in the upstream portion of the O(R), suggesting that the regulatory sequences involved in the replication initiation process have changed through insect evolution.

AT Rich Sequence↗
PubMed1985

In vitro replication of human mitochondrial DNA: accurate initiation at the origin of light-strand synthesis.

Synthesis of human light-strand mitochondrial DNA was accomplished in vitro using DNA primase, DNA polymerase, and other accessory proteins isolated from human mitochondria. Replication begins with the synthesis of primer RNA on a T-rich sequence in the origin stem-loop structure of the template DNA and absolutely requires ATP. A transition from RNA synthesis to DNA synthesis occurs near the base of the stem-loop structure and a potential recognition site for signaling that transition has been identified. The start sites of the in vitro products were mapped at the nucleotide level and were found to be in excellent agreement with those of in vivo nascent light-strand DNA. Isolated human mitochondrial enzymes recognize and utilize the bovine, but not the mouse, origin of light-strand replication.

Animals↗
PubMed2004

Discovery of a major D-loop replication origin reveals two modes of human mtDNA synthesis.

Mammalian mitochondrial DNA (mtDNA) replication has long been considered to occur by asymmetric synthesis of the two strands, starting at the multiple origins of the strand-displacement loop (D-loop). We report the discovery of a major replication origin at position 57 in the D-loop of several human cell lines (HeLa, A549, and 143B.TK-) and immortalized lymphocytes. The nascent chains starting at this origin, in contrast to those initiated at the previously described origins, do not terminate prematurely at the 3' end of the D-loop but proceed well beyond this control point, behaving as "true" replicating strands. This origin is mainly responsible for mtDNA maintenance under steady-state conditions, whereas mtDNA synthesis from the formerly identified D-loop origins may be more important for recovery after mtDNA depletion and for accelerating mtDNA replication in response to physiological demands.

Cell Line↗
PubMed1980

A gamma-like DNA polymerase in spinach chloroplasts.

A DNA polymerase has been extracted from spinach chloroplasts and purified by chromatography on DEAE-cellulose and hydroxyapatite. A great similarity between the purified chloroplast polymerase and the mammalian mitochondrial DNA polymerase gamma was found by several criteria: preference for the synthetic primer-template (dT)12-18 . poly(rA), optimal requirement for Mn2+ (0.1-1.0 mM), KCl (100 mM) and pH (8-9), high relative molecular mass (approximately 105,000), resistance to aphidicolin and inhibition by N-ethylmaleimide. Some peculiar features of the chloroplast DNA polymerase have, however, been noticed. The mammalian DNA polymerase gamma has been suggested to be responsible for the replication of mitochondrial DNA. Thus, both the presence of a gamma-like DNA polymerase in chloroplasts and the similarities between the chloroplast and the mitochondrial DNA (absence of a nucleosomal structure an presence of displacement loops) lead to the suggestion that chloroplast DNA is also replicated by a gamma-like DNA polymerase and that the gamma polymerases present in eukaryotes are, therefore, involved in a strand-displacement DNA synthesis. An alpha-like DNA polymerase activity, present and predominant in crude leaf extracts, was practically absent from purified chloroplast preparations.

Chloroplasts↗
PubMed2007

Impact of assisted reproductive technologies: a mitochondrial perspective of cytoplasmic transplantation.

Many of the assisted reproductive techniques associated with maternal aging, disease states, or implantation failure aim to correct poor developmental capacity. These techniques are highly invasive and require the exchange of nuclear or cytoplasmic material from a donor oocyte to compensate for deficiencies inherent in the affected individual. These techniques are based on the assumption that the cytoplasm of the donor oocyte can effectively substitute the necessary component(s) to enable development to proceed. Several studies have attempted to inject cytoplasm from "normal" (young) donors, into aged eggs, again assuming that beneficial components of the cytoplasm are transferred to restore developmental capacity. These invasive assisted reproduction technology (ART) procedures aim to eliminate chromosomal abnormalities, improve the quality of oocytes deficient in some important cytoplasmic factors necessary for maturation and/or subsequent development, and eliminate maternally inherited diseases (particularly mitochondrial myopathies). However, in order to develop such ART, understanding the processes involving mitochondrial DNA replication and transcription is imperative, as asynchrony between mitochondrial and nuclear genomes may cause problems in mitochondrial function, localization, and biogenesis.

Animals↗
PubMed1983

The effect of retroviral transformation on DNA replication and DNA polymerase-gamma activity in chick embryo fibroblast mitochondria.

The effect of transformation by oncogenic Rous sarcoma viruses on the replication of mitochondrial DNA (mtDNA) in chick embryo fibroblasts (CEF) was investigated, extending our previous report of a three- to five-fold increase in the rate of mtDNA replication, which is strictly linked to the expression of the transformed state, is mitochondria-specific, and is not attributable to virus production per se or different growth rates between normal and transformed CEF. In this paper, in vivo pulse-label and pulse-chase analysis shows an increased specific activity in all the replicative and topological forms of transformed cell mtDNA I, labeled within a 10-min pulse, 30-min chase period, reflecting about the same proportion of total label incorporated into D-loop strands (approximately 9S) relative to full-length closed circular forms (approximately 37S) of mtDNA from both cell types. In contrast to the concomitant changes observed in many other systems with elevated DNA synthesis, neither the estimated intramitochondrial pool size of the labeled DNA precursor (dTTP), nor the total level of the mtDNA-replicating enzyme (mt gamma-polymerase) is increased in the transformed cells. Notably, however, in both cell types the mitochondrial dTTP pools relative to the mtDNA complement are significantly larger than whole-cell pools relative to the nuclear DNA complement, confirming recent reports in HeLa cells. The solubilized mt gamma-polymerases from normal and transformed CEF, respectively, are both precipitated by 50% ammonium sulfate, inhibited by N-ethylmaleimide, have similar sedimentation coefficients, and exhibit optimal activity when poly(rA) . d(pT)10 is used as the template-primer. On the other hand, the transformed cell enzyme demonstrates an altered response to thiol compounds, a decreased tendency to aggregate during sedimentation, and is significantly less tightly attached to the mitochondria than the normal cell enzyme. We conclude that, as a result of transformation, an increased fraction of mtDNA molecules replicate at a given time, and that this increased replication rate in vivo is correlated with the expression of several altered endogenous properties, which possibly include a modified intramitochondrial structural attachment of the mt gamma-polymerase in situ. This experimental system may be well suitable for use in the identification of regulatory factors which function during the replication of the mitochondrial genome in vivo.

Animals↗
PubMed1981

Origin of replication from Xenopus laevis mitochondrial DNA promotes high-frequency transformation of yeast.

A specific fraction of chromosomal DNA from both yeast and a wide variety of other eukaryotes, but not from Escherichia coli, promotes high-frequency transformation in yeast. The plasmids containing these sequences are maintained as extra-chromosomal molecules in transformed cells. These results suggest that similar or identical sequences are used for the initiation of DNA replication in eukaryotes. To test this hypothesis, several foreign eukaryotic DNAs implicated directly or indirectly in the initiation of DNA replication have been examined for their ability to promote autonomous, extrachromosomal replication in yeast. Simian virus 40 DNA, amplified Xenopus laevis ribosomal DNA, X. laevis 5S ribosomal DNA, X. laevis mtDNA, and five different members of the Alu I family of human middle repetitive DNAs were cloned into the vector YIp5 and used to transform yeast. Of these DNAs, only Xenopus mtDNA promoted high-frequency transformation and extrachromosomal maintenance of YIp5 DNA. A 2.2-kilobase EcoRI fragment from the 17.4-kilobase mtDNA molecule was responsible for these activities. This fragment contains the sequence used for the initiation of replication in Xenopus mitochondria.

Animals↗
PubMed2002

The accessory subunit of DNA polymerase gamma is essential for mitochondrial DNA maintenance and development in Drosophila melanogaster.

DNA polymerase gamma, Pol gamma, is the key replicative enzyme in animal mitochondria. The Drosophila enzyme is a heterodimer comprising catalytic and accessory subunits of 125 kDa and 35 kDa, respectively. Both subunits have been cloned and characterized in a variety of model systems, and genetic mutants of the catalytic subunit were first identified in Drosophila, as chemically induced mutations that disrupt larval behavior (tamas). Mutations in the gene encoding the accessory subunit have not yet been described in any organism. Here, we report the consequences of null mutations upon mitochondrial DNA (mtDNA) replication and morphology, cell proliferation, and organismal viability. Mutations in the accessory subunit cause lethality during early pupation, concomitant with loss of mtDNA and mitochondrial mass, and reduced cell proliferation in the central nervous system. Surprisingly, the function of the central nervous system and muscle, as assessed in a locomotion assay, are only marginally affected. This finding is in contrast to our previous findings that disruption in the function of the catalytic subunit causes severe reduction in larval locomotion. We discuss our results in the context of current hypotheses for the function of the accessory subunit in mtDNA replication.

Amino Acid Motifs↗
PubMed1978

Biochemical and electron microscopic characterization of DNA-RNA complexes from HeLa cell mitochondria.

The previous electron microscopic investigations on the occurrence in HeLa cell mitochondria of transcription complexes of mitochondrial DNA [Aloni, Y., and Attardi, G. (1972a), J. Mol. Biol. 70, 363-373] have been extended with the aim of obtaining these complexes in a reasonably pure form for biochemical analysis. By using conditions designed to minimize losses of such structures and any possible contamination by nuclear DNA, it has been shown that a substantial fraction (40 to 50%) of mitochondrial DNA can be isolated from exponentially growing HeLa cells in the form of fastsedimenting complexes with RNA. These complexes have been characterized with respect to density and sedimentation properties, content in newly synthesized RNA, stability of the association of RNA with DNA, presence of different forms of mitochondrial DNA, and electron microscopic appearance. The properties of these complexes, as well as the results of reconstruction experiments, strongly suggest that the majority of such structures represent true transcriptional intermediates. The occurrence in this fraction of replicating or newly replicated mitochondrial DNA molecules has been observed. Although the presence of single-stranded DNA segments makes the replicative intermediates particularly susceptible to aggregation with free RNA, electron microscopic observations point to the possibility that these intermediates may be recruited for transcription.

DNA, Mitochondrial↗
PubMed1994

Mitochondrial minicircle DNA supports plasmid replication and maintenance in nuclei of Trypanosoma brucei.

In a search for trypanosome DNA sequences that permit replication and stable maintenance of extrachromosomal elements, a 1-kilobase-pair (kbp) fragment from a mitochondrial kinetoplast DNA (kDNA) minicircle of Trypanosoma brucei was isolated and characterized. The plasmid pTbo-1, carrying the kDNA element, is maintained in T. brucei as a supercoiled concatemer containing approximately seven to nine pTbo-1 monomer units (5.6 kbp each) in a head-to-tail orientation. The concatemer is found in approximately one copy per cell when procyclic trypanosomes are cultured in the presence of 100 micrograms of hygromycin per ml; however, in the absence of continuous hygromycin selection, the plasmid is lost from the population with a t1/2 of approximately 8.7 days (17 cell generations). A second unrelated kDNA minicircle was also able to serve as an autonomously replicating sequence (ARS) element in T. brucei, suggesting that this is a general property of kDNA minicircles. Replication of mitochondrial DNA in the nucleus may be due to either a specific consensus sequence (such as in yeast ARS elements) or nonspecific sequence characteristics (such as the degree of A&T-richness or bent DNA).

Animals↗
PubMed1990

Mapping light strand transcripts near the origin of replication of Xenopus laevis mitochondrial DNA.

Transcription of the light strand of Xenopus laevis mitochondrial DNA initiates at two promoters located approximately 350 to 450 nucleotides upstream from the 5' ends of major D-loop DNA strands. Small RNAs within this region have been mapped by blot hybridization, primer extension and S1 nuclease protection methods. The results reveal that the large majority of RNAs within this region have 3' termini located at a sequence element, designated CSB 2, that is conserved in sequence and position in Xenopus, mouse, rat and human mtDNA. However, the X. laevis CSB 2 appears to be a site of RNA processing only, since RNA-to-DNA transitions are not detectable at this site. RNAs containing sequences downstream of CSB 2 are extremely rare. A significant fraction of these RNAs are processed by cleavage at a site just upstream of the most predominant 5' ends of D-loop DNAs. We suggest that RNA processing at this site may play a role in priming mtDNA replication.

Animals↗
PubMed2004

Functional analysis of subcellular localization and protein-protein interaction sequences in the essential DNA ligase I protein of fission yeast.

DNA ligase I (Lig I) has key roles in chromosomal DNA replication and repair in the eukaryotic cell nucleus. In the budding yeast Saccharomyces cerevisiae the Lig I enzyme Cdc9p is also required for mitochondrial DNA replication and repair. In this report, dual nuclear-mitochondrial localization is demonstrated to be a property of the essential Lig I enzyme Cdc17 from the distantly related fission yeast Schizosaccharomyces pombe. Expression of nuclear and mitochondrial forms of Cdc17 from separate genes shows that, whereas expression of either protein alone is insufficient to restore viability to cells lacking endogenous Cdc17, co-expression restores full viability. In the nucleus, Lig I interacts with the sliding clamp proliferating cell nuclear antigen (PCNA) via a conserved PCNA interacting sequence motif known as a PIP box. Deletion of the PIP motif from the N-terminus of the nuclear form of Cdc17 fails to abolish Cdc17 function, indicating that PCNA binding by Cdc17 is not an absolute requirement for completion of S-phase.

Amino Acid Motifs↗
PubMed1998

AZT decreases rat myocardial cytochrome oxidase activity and increases beta-myosin heavy chain content.

AZT, a widely-utilized drug for the treatment of HIV infection, inhibits the polymerase responsible for mitochondrial DNA replication (mtDNA). The aim of this study was to assess myocardial alterations caused by this action. Ventricular muscle from rats treated for > or = 35 days with 1 mg/ml of AZT in their drinking water was analysed for cytochrome oxidase activity and the content of mRNAs for the nuclear-encoded cytochrome oxidase (COX) subunit VIc and the mitochondrial-encoded COX subunit III. In addition contractile protein expression was assessed by examining mRNA levels for alpha- and beta-myosin heavy chains (MHC). Changes in MHC mRNA levels were correlated with changes in alpha- and beta-MHC proteins and changes in myofibrillar ATPase activity. Results show that AZT caused a reduction in COX activity, COX subunit III mRNA, and mtDNA levels. There was no decrease in the COX subunit VIc mRNA. MHC expression was altered such that the relative content of beta-MHC protein and mRNA were increased. Accumulation of beta-MHC was reflected in the reduction of myofibrillar ATPase activity at pCa values of 5.875 and 6.125. These data demonstrate that AZT induces a reorganization of cardiac gene expression indicative of changes in cardiac contractile properties. The observed decreases in mtDNA levels along with mRNA for a mitochondrial-encoded protein and COX activity is consistent with the postulated mechanism whereby AZT induces a myopathy by diminishing mtDNA replication.

Animals↗
PubMed1995

A test of the transcription model for biased inheritance of yeast mitochondrial DNA.

Two strand-specific origins of replication appear to be required for mammalian mitochondrial DNA (mtDNA) replication. Structural equivalents of these origins are found in the rep sequences of Saccharomyces cerevisiae mtDNA. These striking similarities have contributed to a universal model for the initiation of mtDNA replication in which a primer is created by cleavage of an origin region transcript. Consistent with this model are the properties of deletion mutants of yeast mtDNA ([rho-]) with a high density of reps (HS [rho-]). These mutant mtDNAs are preferentially inherited by the progeny resulting from the mating of HS [rho-] cells with cells containing wild-type mtDNA ([rho+]). This bias is presumed to result from a replication advantage conferred on HS [rho-] mtDNA by the high density of rep sequences acting as origins. To test whether transcription is indeed required for the preferential inheritance of HS [rho-] mtDNA, we deleted the nuclear gene (RPO41) for the mitochondrial RNA polymerase, reducing transcripts by at least 1000-fold. Since [rho-] genomes, but not [rho+] genomes, are stable when RPO41 is deleted, we examined matings between HS [rho-] and neutral [rho-] cells. Neutral [rho-] mtDNAs lack rep sequences and are not preferentially inherited in [rho-] x [rho+] crosses. In HS [rho-] x neutral [rho-] matings, the HS [rho-] mtDNA was preferentially inherited whether both parents were wild type or both were deleted for RPO41. Thus, transcription from the rep promoter does not appear to be necessary for biased inheritance. Our results, and analysis of the literature, suggest that priming by transcription is not a universal mechanism for mtDNA replication initiation.

Cell Nucleus↗