Mitochondrial Replication Research Articles

Effect of length variations at nucleotide positions 303–315 in human mitochondrial DNA on transcription termination

In addition to stabilizing the RNA–DNA hybrid, conserved sequence block (CSB) II, which is located at nucleotides 299–315 on mitochondrial DNA, relates with the transcription termination for initiation of heavy strand synthesis in human mitochondrial replication. Due to length polymorphisms at nucleotides 303–315, individuals contain homo- or heteroplasmic profiles with length variants from C 5TC 6 to C 15TC 6 or from C 9 to C 13. Using in vitro transcription with templates containing these variations, we demonstrated that the production of prematurely terminated (PT) transcripts depends on the 303–315 sequences, and that longer templates result in relatively higher levels of PT transcripts. The 3′ ends of PT transcripts were observed within or downstream of CSB II. Termination positions downstream of nucleotide 303 were shifted upstream by longer variations, but not shifted by shorter variations. We found that length variations between 303 and 315 generate quantitative and qualitative differences in PT transcripts.

Characterization of mitochondrial replication and transcription control during rat early development in vivo and in vitro

In vitro culture (IVC), used in assisted reproductive technologies, is a major environmental stress on the embryo. To evaluate the effect of IVC on mitochondrial transcription and the control of mtDNA replication, we measured the mtDNA copy number and relative amount of mRNA for mitochondrial-related genes in individual rat oocytes, zygotes and embryos using real-time PCR. The average mtDNA copy number was 147 600 (+/-3000) in metaphase II oocytes. The mtDNA copy number was stable throughout in vivo early development and IVC induced an increase in mtDNA copy number from the 8-cell stage onwards. Gapd mRNA levels vary during early development and IVC did not change the patterns of these housekeeping gene transcripts. Polrmt mRNA levels did not vary during early development up to the morula stage but increased at the blastocyst stage. IVC induced the up-regulation of Polrmt mRNA, one of the key genes regulating mtDNA transcription and replication, at the blastocyst stage. An increase in mt-Nd4 mRNA preceded the blastocyst-related event observed in nuclear-encoded Gapd and Polrmt, suggesting that the expression of mitochondrial encoded genes is controlled differently from nuclear encoded genes. We conclude that the IVC system can perturb mitochondrial transcription and the control of mtDNA replication in rat embryos. This perturbation of mtDNA regulation may be responsible for the abnormal physiology, metabolism and viability of in vitro-derived embryos.

The accessory subunit B of DNA polymerase is required for mitochondrial replisome function

The mitochondrial replication machinery in human cells includes the DNA polymerase γ holoenzyme and the TWINKLE helicase. Together, these two factors form a processive replication machinery, a replisome, which can use duplex DNA as template to synthesize long stretches of single-stranded DNA. We here address the importance of the smaller, accessory B subunit of DNA polymerase γ and demonstrate that this subunit is absolutely required for replisome function. The duplex DNA binding activity of the B subunit is needed for coordination of POLγ holoenzyme and TWINKLE helicase activities at the mtDNA replication fork. In the absence of proof for direct physical interactions between the components of the mitochondrial replisome, these functional interactions may explain the strict interdependence of TWINKLE and DNA polymerase γ for mitochondrial DNA synthesis. Furthermore, mutations in TWINKLE as well as in the catalytic A and accessory B subunits of the POLγ holoenzyme, may cause autosomal dominant progressive external ophthalmoplegia, a disorder associated with deletions in mitochondrial DNA. The crucial importance of the B subunit for replisome function may help to explain why mutations in these three proteins cause an identical syndrome.

287 ACTIVE MITOCHONDRIA ARE PRESENT IN MOUSE AND MONKEY EMBRYONIC STEM CELL LINES

The inner cell mass (ICM) of the blastocyst develops into the fetus after uterine implantation. Prior to implantation, ICM cells synthesize ATP by glycolytic reactions. We now report that cells of the ICM in 3.5-day-old mouse embryos have too few mitochondria to be visualized with either Mitotracker red (active mitochondria) or an antibody against complex I of OXPHOS. By comparison, all of the surrounding trophectoderm cells reveal numerous mitochondria throughout their cytoplasm. It has largely been assumed that embryonic stem (ES) stem cells derived from the ICM also have few mitochondria, and that replication of mitochondria in the ES cells does not begin until they commence differentiation. We further report that mouse E14 ES cells and monkey ORMES 7 ES cells have considerable numbers of active mitochondria when cultured under standard conditions, i.e., 5% CO2 in air. Both the mouse E14 and monkey ES cell lines expressed two markers of undifferentiated cells, Oct-4 and SSEA-4, and monkey ES cells expressed the undifferentiated cell marker Nanog; however, Oct-4 is nonspecific in monkey ES cells because trophectoderm also expresses this marker, unlike in mice. Ninety-nine percent of the E14 cells examined, and 100% of the ORMES 7 cells, have a visible mitochondrial mass when stained with either Mitoracker red or with an antibody against OXPHOS complex I. The ATP content in the mouse E14 cells (4.13 pmoles ATP/cell) is not significantly different (P = 0.76) from that in a mouse fibroblast control (3.75 pmoles ATP/cell). Cells of the monkey ORMES 7 cell line had 61% of the ATP/cell content (7.55 pmoles ATP/cell) compared to the monkey fibroblast control (12.38 pmoles ATP/cell). Both cell lines expressed two proteins believed to indicate competence of mitochondria to replicate: PolG, the polymerase used to replicate the mitochondrial genome, and TFAM, a nuclear-encoded transcription factor reported to regulate several aspects of mitochondrial function. Both proteins were found to co-localize in the mitochondria. We conclude that when the ICMs are isolated from blastocysts and used to establish these two ES cell lines in cell culture, mitochondrial biosynthesis is activated.

Skeletal Muscle of Female Rats Exhibit Higher Mitochondrial Mass and Oxidative-Phosphorylative Capacities Compared to Males

The effect of gender and caloric restriction on mitochondrial content and oxidative-phosphorylative capacities has been investigated in rat gastrocnemius muscle. Muscle protein, mitochondrial protein and DNA contents, enzymatic activities of mitochondrial oxidative and phosphorylative system, mitochondrial antioxidant enzymes, protein levels of complex IV (subunit I and IV) and ATPase, and the gene and protein expression of mitochondrial transcription factor A (TFAM), involved in mitochondrial replication and transcription, were measured in rats of both genders fed ad libitum and subjected to three months of 40% caloric restriction. Compared to males, gastrocnemius muscle of female rats showed higher mitochondrial DNA and protein contents, TFAM protein level, oxidative and phosphorylative machinery and activities, and glutathione peroxidase activity. In conclusion, the present data show a clear gender dimorphism in rat muscle mitochondrial features, which could explain the higher facility of females to adapt to altered metabolic energy situations.

Mitochondrial Drug Targets in Apicomplexan Parasites

In evolutionary terms, mitochondria in apicomplexan parasites appear to be "relicts-in-the-making": they possess the smallest mitochondrial genomes known, encoding only three proteins, and in one genus, Cryptosporidium, the genome is eliminated altogether. Several features of mitochondrial physiology provide validated or potential targets for antiparasitic drugs. Atovaquone, a broad spectrum antiparasitic drug, selectively inhibits mitochondrial electron transport at the cytochrome bc(1) complex and collapses mitochondrial membrane potential. Recent investigations using model systems provide important insights into the mechanism of action for this drug, which may prove valuable for development of other selective inhibitors of mitochondrial electron transport. Although mitochondria do not appear to be a source of ATP during the erythrocytic stages in Plasmodium species, they do serve other critical functions, including the assembly of iron-sulfur clusters and various other biosynthetic processes depending on the species. To serve these metabolic functions, parasites need to maintain the apparatus for mitochondrial genome replication, repair, recombination, transcription, and translation, components of which are encoded in the nucleus and imported into the mitochondrion. Several unusual aspects of the components of this apparatus are coming to light through genome sequence analyses, and could provide potential targets for antiparasitic drug discovery and development.

Regulated Mitochondrial DNA Replication During Oocyte Maturation Is Essential for Successful Porcine Embryonic Development

Cellular ATP is mainly generated through mitochondrial oxidative phosphorylation, which is dependent on mitochondrial DNA (mtDNA). We have previously demonstrated the importance of oocyte mtDNA for porcine and human fertilization. However, the role of nuclear-encoded mitochondrial replication factors during oocyte and embryo development is not yet understood. We have analyzed two key factors, mitochondrial transcription factor A (TFAM) and polymerase gamma (POLG), to determine their role in oocyte and early embryo development. Competent and incompetent oocytes, as determined by brilliant cresyl blue (BCB) dye, were assessed intermittently during the maturation process for TFAM and POLG mRNA using real-time RT-PCR, for TFAM and POLG protein using immunocytochemistry, and for mtDNA copy number using real-time PCR. Analysis was also carried out following treatment of maturing oocytes with the mtDNA replication inhibitor, 2',3'-dideoxycytidine (ddC). Following in vitro fertilization, preimplantation embryos were also analyzed. Despite increased levels of TFAM and POLG mRNA and protein at the four-cell stage, no increase in mtDNA copy number was observed in early preimplantation development. To compensate for this, mtDNA appeared to be replicated during oocyte maturation. However, significant differences in nuclear-encoded regulatory protein expression were observed between BCB(+) and BCB(-) oocytes and between untreated oocytes and those treated with ddC. These changes resulted in delayed mtDNA replication, which correlated to reduced fertilization and embryonic development. We therefore conclude that adherence to the regulation of the timing of mtDNA replication during oocyte maturation is essential for successful embryonic development.

MTERF3, the most conserved member of the mTERF-family, is a modular factor involved in mitochondrial protein synthesis

The MTERF-family is a wide family of proteins identified in Metazoa and plants which includes the known mitochondrial transcription termination factors. With the aim to shed light on the function of MTERF-family members in Drosophila, we performed the cloning and characterization of D-MTERF3, a component of the most conserved group of this family. D-MTERF3 is a mitochondrial protein of 323 amino acids. Sequence analysis in seven different organisms showed that the protein contains five conserved “mTERF-motifs”, three of which include a leucine zipper-like domain. D-MTERF3 knock-down, obtained by RNAi in D.Mel-2 cells, did not affect mitochondrial replication and transcription. On the contrary, it decreased to a variable extent the rate of labelling of about half of the mitochondrial polypeptides, with ND1 being the most affected by D-MTERF3 depletion. These results indicate that D-MTERF3 is involved in mitochondrial translation. This role, likely based on protein–protein interactions, may be exerted either through a direct interaction with the translation machinery or by bridging the mitochondrial transcription and translation apparatus.

Mitochondrial DNA copy number is regulated by cellular proliferation: A role for Ras and p66 Shc

The abundance of mitochondria is regulated by biogenesis and division. These processes are controlled by cellular factors, given that, for example, mitochondria have to replicate their DNA prior to cell division. However, the mechanisms that allow a synchronization of cell proliferation with mitochondrial genome replication are still obscure. We report here our investigations on the role of proliferation and the contribution of Ras and p66 Shc in the regulation of mitochondrial DNA copy number. Ras proteins mediate a variety of receptor-transduced mitogenic signals and appear to play an essential role in the cellular response to growth factors. P66 Shc is a genetic determinant of life span in mammals and has been implicated in the regulation of receptor signaling and various mitochondrial functions. First, we confirmed previous reports showing that mitochondrial DNA is replicated during a specific phase of the cell cycle (the pre-S phase) and provided novel evidences that this process is regulated by mitogenic growth factors. Second, we showed that mitochondrial DNA replication is activated following Ras-induced cellular hyper-proliferation. Finally, we showed that p66 Shc expression induces mitochondrial DNA replication, both in vitro and in vivo. We suggest that mitochondria are target of intracellular signaling pathways leading to proliferation, involving Ras and p66 Shc, which might function to integrate cellular bio-energetic requirements and the inheritance of mitochondrial DNA in a cell cycle-dependent manner.

Aberrant nucleo-cytoplasmic cross-talk results in donor cell mtDNA persistence in cloned embryos.

Mitochondrial DNA is an extranuclear genome normally maternally inherited through the oocyte. However, the use of nuclear transfer can result in both donor cell and recipient oocyte mitochondrial DNA persisting through to blastocyst and being transmitted to the offspring. The degree of donor mitochondrial DNA transmission appears to be random and currently no evidence exists to explain this phenomenon. To determine whether this is a dilution factor or directly related to the transcriptional status of the donor cell in respect of mitochondrial DNA transcription factors, we have generated sheep nuclear transfer embryos using donor cells: (1) possessing their full mitochondrial DNA complement, (2) those partially depleted, and (3) those depleted but containing residual levels. For each donor type, donor mitochondrial DNA persisted in some blastocysts. It is evident from the donor cells used that nuclear-encoded mitochondrial DNA transcription and replication factors persist even after mitochondrial DNA depletion, as do transcripts for some of the mitochondrial-encoded genes. These cells are therefore still programmed to drive mitochondrial DNA replication and transcription. In nuclear transfer-derived embryos, we have observed the persistence of these nuclear-encoded mitochondrial DNA transcription and replication factors but not in those embryos generated through in vitro fertilization. Consequently, nucleo-mitochondrial interaction following nuclear transfer is out of sequence as the onset of mitochondrial replication is a postimplantation event.

Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20

Fertilizable mammalian oocytes are arrested at the second meiotic metaphase (mII) by the cyclinB-Cdc2 heterodimer, maturation promoting factor (MPF). MPF is stabilized via the activity of an unidentified cytostatic factor (CSF), thereby suspending meiotic progression until fertilization. We here present evidence that a conserved 71 kDa mammalian orthologue of Xenopus XErp1/Emi2, which we term endogenous meiotic inhibitor 2 (Emi2) is an essential CSF component. Depletion in situ of Emi2 by RNA interference elicited precocious meiotic exit in maturing mouse oocytes. Reduction of Emi2 released mature mII oocytes from cytostatic arrest, frequently inducing cytodegeneration. Mos levels autonomously declined to undetectable levels in mII oocytes. Recombinant Emi2 reduced the propensity of mII oocytes to exit meiosis in response to activating stimuli. Emi2 and Cdc20 proteins mutually interact and Cdc20 ablation negated the ability of Emi2 removal to induce metaphase release. Consistent with this, Cdc20 removal prevented parthenogenetic or sperm-induced meiotic exit. These studies show in intact oocytes that the interaction of Emi2 with Cdc20 links activating stimuli to meiotic resumption at fertilization and during parthenogenesis in mammals.

Mitochondrial replication origin stability and propensity of adjacent tRNA genes to form putative replication origins increase developmental stability in Lizards

Secondary structure stability of mitochondrial origins of light-strand replication (OL) presumably reduces delayed formation of light-strand initiating replication forks on the heavy strand. Delayed replication initiation prolongs single strandedness of the heavy strand. More mutations accumulate during the prolonged time spent single stranded. Presumably, delayed replication initiation and excess mutations affect mitochondrial biochemical processes and ultimately morphological outcomes of development at the whole-organism level. This predicts that developmental stability increases with OL secondary structure stability and with formation of OL-like structures by the five tRNA genes flanking recognized OLs. Stable OLs and high percentages of OL-resembling secondary structures of adjacent tRNA genes (predicted by Mfold) correlate positively with developmental stability in three lizard families (Anguidae, Amphisbaenidae, and Polychrotidae). Accounting for effects of the regular OL, Sfold-predicted OL-like propensity of the entire tRNA gene cluster (not of individual genes) correlates with increased developmental stability in Anguidae, also across the entire free-energy range of Boltzmann's distribution of secondary structures. In the fossorial Amphisbaenidae, the OL-like structure-forming propensity of tRNA genes correlates positively with developmental stability for the distribution's sub-optimally stable regions, and negatively for its optimally stable regions, suggesting the thermoregulated functioning of OL vs. flanking tRNA genes as replication origins. Results for polychrotid tRNA genes are intermediate. Anguid tRNA genes possibly function in addition to the regular OL. Mitochondrial tRNA genes may thus frequently acquire and lose the alternative OL function, without sequence (gene) duplication and loss of their primary function.

Bacteriophage origins of mitochondrial replication and transcription proteins

Mounting evidence suggests that key components of the mitochondrial transcription and replication apparatus are derived from the T-odd lineage of bacteriophage rather than from an alpha-Proteobacterium, as the endosymbiont hypothesis would predict. We propose that several mitochondrial replication genes were acquired together from an ancestor of T-odd phage early in the evolution of the eukaryotic cell, at the time of the mitochondrial endosymbiosis. We further propose that at a later stage the single-subunit RNA polymerase, originally acquired for mitochondrial DNA replication, was co-opted to serve in mitochondrial transcription.

Mitochondrial Genome Instability in AML Is Associated with Reduction in the Mitochondrial Replication.

Genetic changes in mitochondrial DNA (mtDNA) have been hypothesized more widely to play important roles in senescence, autoimmune disease and malignancy because of a paucity of introns and limited repair mechanisms. Malfunction of mismatch repair genes produces genome instability which plays an important role in the development of human cancers. The mtDNA markers for mitochondrial genome instability (mtGI) were point mutations, insertions, deletions and length changes in homopolymeric nucleotide tracts. We investigated the mtGI in AML cells and its effect to alteration in mtDNA copy number. Forty-eight matched AML bone marrow and buccal mucosa samples, and blood samples from 57 control subjects were collected after receiving Institutional Review Board approval and informed consent. We directly sequenced the control region, the tRNA leucine 1 gene plus a part of NADH dehydrogenase (ND)1 and cytochrome b (CYTB) of mtDNA. In an attempt to investigate mtGI, we carried out a qualitative and quantitative profiling mtDNA length heteroplasmies of six mtGIs (np 303–315 poly C, np 16184–16193 poly C, np 514–511 CA repeats, np 3566–3572 poly C, np 12385–12391 poly C and np 12418–12426 poly A) using a size-based PCR product separation by capillary electrophoresis (ABI Prism Genotyper version 3.1). Length heteroplasmy was further confirmed by cloning and sequencing. Quantitative analysis of mtDNA molecules was performed using the QuantiTect SYBR Green PCR kit (Qiagen). In the current study, we detected a large number of polymorphisms as well as new mtDNA variants. A total of 606 mtDNA sequence variants were identified. Among these, 15 mtDNA variants were identified as novel mutations that were absent from corresponding buccal mucosa, control subjects and established mtDNA polymorphism databases. In the control region, we found two types of mtDNA alterations - base substitutions and small deletions/insertions as well as the length heteroplasmies in the np 303 to 315 poly-C, np 16184 to 16193 poly-C and 514–515 CA repeats. Seven patients (15%) had leukemia cell-specific mtDNA substitution mutations in the ND1 and CYTB genes. Somatic mtDNA control region mutations found in this study preferentially altered known mtDNA regulatory elements. AML cells had about a two-fold decrease in mtDNA copy number compared with the results from control subjects (63 x 106 molecules/ul ± 23 x 106 vs 122 x 106 molecules/ul ± 73 x 106). Our results are consistent with a recent observation that carcinogenesis in the liver, kidney and lung involves a decrease of the cellular mitochondrial content and decreased mtDNA copy number (Mutat Res 2004; 547:71–78). In conclusion, mtGI including point mutations, length changes (insertions or deletions) in homopolymeric tracts commonly occurred in AML cells and reduction in mtDNA copy number may result from either mtDNA control region mutations or impairment of mitochondrial biogenesis. These findings suggest that biogenesis of mitochondria is repressed in the leukemogenesis process of the human hematopoietic tissue.

On the relevance of mitochondrial fusions for the accumulation of mitochondrial deletion mutants: A modelling study

The molecular mechanisms underlying the aging process are still unclear, but the clonal accumulation of mitochondrial deletion mutants is one of the prime candidates. An important question for the mitochondrial theory of aging is to discover how defective organelles might be selected at the expense of wild-type mitochondria. We propose that mitochondrial fission and fusion events are of critical importance for resolving this apparent contradiction. We show that the occurrence of fusions removes the problems associated with the idea that smaller DNA molecules accumulate because they replicate in a shorter time--the survival of the tiny (SOT) hypothesis. Furthermore, stochastic simulations of mitochondrial replication, mutation and degradation show that two important experimental findings, namely the overall low mosaic pattern of oxidative phosphorylation (OXPHOS) impaired cells in old organisms and the distribution of deletion sizes, can be reproduced and explained by this hypothesis. Finally, we make predictions that can be tested experimentally to further verify our explanation for the age-related accumulation of mitochondrial deletion mutants.

Association between polymorphisms in the nuclear respiratory factor 1 gene and type 2 diabetes mellitus in the Korean population

Dysfunction in mitochondrial oxidative phosphorylation plays a central role in insulin resistance and type 2 diabetes. Nuclear respiratory factor 1 (NRF1) is a transcription factor that acts on nuclear genes encoding respiratory subunits and components of the mitochondrial transcription and replication machinery. Thus, we investigated its genetic association with type 2 diabetes. The NRF1 gene was sequenced to identify polymorphisms in 24 Korean DNA samples and then common variants were genotyped in 766 patients with type 2 diabetes and 303 non-diabetic subjects. Twelve single nucleotide polymorphisms and one insertion/deletion polymorphism were identified. Six common variants among them were genotyped in a larger study. Although three individual polymorphisms appeared to be associated with type 2 diabetes (g.-46350insdel A, g.+141G>T and g.+54529A>G), the effects were only marginal. However, a haplotype (H2) was associated with a decreased risk of type 2 diabetes and another haplotype (H4) was associated with an increased risk of type 2 diabetes (p values for the Haplo. Score test were 0.009 and 0.004, respectively). We demonstrated that two common haplotypes of NRF1 gene are associated with type 2 diabetes in the Korean population.

Deficiency of the ADP-Forming Succinyl-CoA Synthase Activity Is Associated with Encephalomyopathy and Mitochondrial DNA Depletion

The mitochondrial DNA (mtDNA) depletion syndrome is a quantitative defect of mtDNA resulting from dysfunction of one of several nuclear-encoded factors responsible for maintenance of mitochondrial deoxyribonucleoside triphosphate (dNTP) pools or replication of mtDNA. Markedly decreased succinyl-CoA synthetase activity due to a deleterious mutation in SUCLA2, the gene encoding the beta subunit of the ADP-forming succinyl-CoA synthetase ligase, was found in muscle mitochondria of patients with encephalomyopathy and mtDNA depletion. Succinyl-CoA synthetase is invariably in a complex with mitochondrial nucleotide diphosphate kinase; hence, we propose that a defect in the last step of mitochondrial dNTP salvage is a novel cause of the mtDNA depletion syndrome.

Molecular control of mitochondrial function in preimplantation mouse embryos

Mitochondria play a key role in a number of physiological events during all stages of life, including the very first stages following fertilization. It is, therefore, important to understand the mechanisms controlling mitochondrial activity during early embryogenesis to determine their role in development outcome. The objective of this study was to investigate the molecular control of mitochondrial transcription and mitochondrial DNA (mtDNA) replication in mouse preimplantation embryos. We estimated the mtDNA copy number and characterized the expression patterns of two mitochondrial genes and several nuclear genes that encode mitochondrial transcription and replication factors throughout preimplantation development. Mitochondrial gene transcripts were present in larger quantities in morula and blastocyst stage embryos relative to other stages. A significant increase in the amount of mRNA for nuclear genes encoding mtDNA transcription factors was observed in eight-cell stage embryos. Although a similar increase in the mRNA levels of nuclear genes encoding mtDNA replication factors was observed in morula and blastocyst stage embryos, the number of mtDNA molecules remained stable during preimplantation stages, suggesting that nuclear-encoded mitochondrial transcription factors are involved in the regulation of mtDNA transcription during early development. Although transcripts of replication factors are abundant at the morula and blastocyst stage, mtDNA replication did not occur until the blastocyst stage, suggesting that the inhibition of mtDNA replication is controlled at the post-transcriptional level during early embryogenesis.

Analysis of Proteome Bound to D‐Loop Region of Mitochondrial DNA by DNA‐Linked Affinity Chromatography and Reverse‐Phase Liquid Chromatography/Tandem Mass Spectrometry

Mitochondrial dysfunction has been suggested as a causal factor for insulin resistance and diabetes. Previously we have shown a decrease of mitochondrial DNA (mtDNA) content in tissues of diabetic patients. The mitochondrial proteins, which regulate the mitochondrial biogensis, including transcription and replication of mtDNA, are encoded by nuclear DNA. Despite the potential function of the proteins bound to the D-loop region of mtDNA in regulating mtDNA transcription/replication, only a few proteins are known to bind the D-loop region of mtDNA. The functional association of these known proteins with insulin resistance is weak. In this study, we applied proteomic analysis to identify a group of proteins (proteome) that physically bind to D-loop DNA of mtDNA. We amplified D-loop DNA (1.1 kb) by PCR and conjugated the PCR fragments to CNBr-activated sepharose. Mitochondria fractions were isolated by both differential centrifugation and Optiprep-gradient ultracentrifugation. The D-loop DNA binding proteome fractions were enriched via this affinity chromatography and analyzed by SDS-PAGE. The proteins on the gel were transferred onto PVDF membrane and the peptide sequences of each band were subsequently analyzed by capillary reverse-phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS). We identified many D-loop DNA binding proteins, including mitochondrial transcription factor A (mtTFA, Tfam) and mitochondrial single-stranded DNA binding protein (mtSSBP) which were known to bind to mtDNA. We also report the possibility of novel D-loop binding proteins such as histone family proteins and high-mobility group proteins.

Bidirectional Replication Initiates at Sites Throughout the Mitochondrial Genome of Birds

Analysis of mitochondrial replication intermediates of Gallus gallus on fork-direction gels indicates that replication occurs in both directions around circular mitochondrial DNA. This finding was corroborated by a study of chick mitochondrial DNA on standard neutral two-dimensional agarose gels, which yielded archetypal initiation arcs in fragments covering the entire genome. There was, however, considerable variation in initiation arc intensity. The majority of initiation events map to regions flanking the major non-coding region, in particular the NADH dehydrogenase subunit 6 (ND6) gene. Initiation point mapping of the ND6 gene identified prominent free 5' ends of DNA, which are candidate start sites for DNA synthesis. Therefore we propose that the initiation zone of G. gallus mitochondrial DNA encompasses most, if not all, of the genome, with preferred initiation sites in regions flanking the major non-coding region. Comparison with mammals suggests a common mechanism of initiation of mitochondrial DNA replication in higher vertebrates.