Mitochondrial telomere-binding protein from Candida parapsilosis suggests an evolutionary adaptation of a nonspecific single-stranded DNA-binding protein

The mitochondrial genome in a number of organisms is represented by linear DNA molecules with defined terminal structures. The telomeres of linear mitochondrial DNA (mtDNA) of yeast Candida parapsilosis consist of tandem arrays of large repetitive units possessing single-stranded 5' extension of about 110 nucleotides. Recently we identified the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of C. parapsilosis linear mtDNA and protects it from attack by various DNA-modifying enzymes (Tomáska, L'., Nosek, J., and Fukuhara, H. (1997) J. Biol. Chem. 272, 3049-3059). Here we report the isolation of MTP1, the gene encoding mtTBP of C. parapsilosis. Sequence analysis revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded DNA-binding proteins that nonspecifically bind to single-stranded DNA with high affinity. Recombinant mtTBP displays a preference for the telomeric 5' overhang of C. parapsilosis mtDNA. The heterologous expression of a mtTBP-GFP fusion protein resulted in its localization to the mitochondria but was unable to functionally substitute for the loss of the S. cerevisiae homologue Rimlp. Analysis of the MTP1 gene and its translation product mtTBP may provide an insight into the evolutionary origin of linear mitochondrial genomes and the role it plays in their replication and maintenance.     

Extragenomic double-stranded DNA circles in yeast with linear mitochondrial genomes: potential involvement in telomere maintenance

Although the typical mitochondrial DNA (mtDNA) is portrayed as a circular molecule, a large number of organisms contain linear mitochondrial genomes classified by their telomere structure. The class of mitochondrial telomeres identified in three yeast species, Candida parapsilosis, Pichia philodendra and Candida salmanticensis, is characterized by inverted terminal repeats each consisting of several tandemly repeating units and a 5' single-stranded extension. The molecular mechanisms of the origin, replication and maintenance of this type of mitochondrial telomere remain unknown. While studying the replication of linear mtDNA of C.parapsilosis by 2-D gel electrophoresis distinct DNA fragments composed solely of mitochondrial telomeric sequences were detected and their properties were suggestive of a circular conformation. Electron microscopic analysis of these DNAs revealed the presence of highly supertwisted circular molecules which could be relaxed by DNase I. The minicircles fell into distinct categories based on length, corresponding to n x 0.75 kb (n = 1-7). Similar results were obtained with two other yeast species (P.philodendra and C. salmanticensis) which possess analogous telomeric structure.     

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.     

Electron microscopic analysis supports a dual role for the mitochondrial telomere-binding protein of Candida parapsilosis

Linear mitochondrial genomes exist in several yeast species which are closely related to yeast that harbor circular mitochondrial genomes. Several lines of evidence suggest that the conversion from one form to another occurred accidentally through a relatively simple mechanism. Previously, we (L.T. & J.N.) reported the identification of the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of Candida parapsilosis linear mtDNA, and sequence analysis of the corresponding nuclear gene MTP1 revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded (ss) DNA-binding (SSB) proteins. In this study, the DNA-binding properties of mtTBP in vitro and in vivo were analyzed by electron microscopy (EM). When M13 ssDNA was used as a substrate, mtTBP exhibited similar DNA binding characteristics as human mitochondrial SSB: mtTBP formed protein globules along the DNA substrate, and the bound proteins were randomly distributed, indicating that the binding of mtTBP to M13 ssDNA is not highly cooperative. EM analysis demonstrated that mtTBP is able to recognize the 5' single-stranded telomeric overhangs in their natural context. Using isopycnic centrifugation of mitochondrial lysates of C. papsilosis we show that mtTBP is a structural part of mitochondrial nucleoids of C. parapsilosis and is predominantly bound to the mitochondrial telomeres. These data support a dual role of mtTBP in mitochondria of C. parapsilosis, serving both as a typical mitochondrial SSB and as a specific component of the mitochondrial telomeric chromatin.     

Mitochondrial single-stranded DNA-binding proteins: in search for new functions

During the evolution of the eukaryotic cell, genes encoding proteins involved in the metabolism of mitochondrial DNA (mtDNA) have been transferred from the endosymbiont into the host genome. Mitochondrial single-stranded DNA-binding (mtSSB) proteins serve as an excellent argument supporting this aspect of the endosymbiotic theory. The crystal structure of the human mtSSB, together with an abundance of biochemical and genetic data, revealed several exciting features of mtSSB proteins and enabled a detailed comparison with their prokaryotic counterparts. Moreover, identification of a novel member of the mtSSB family, mitochondrial telomere-binding protein of the yeast Candida parapsilosis, has raised interesting questions regarding mtDNA metabolism and evolution.     

The telomeres of the linear mitochondrial DNA of tetrahymena thermophila consist of 53 bp tandem repeats

We have cloned and sequenced the telomeric DNA of the linear mitochondrial DNA (mtDNA) of T. thermophila BVII. The mtDNA telomeres consist of a 53 bp sequence tandemly repeated from 4 to 30 times, with most molecules having 15 ± 4 repetitions. The previously recognized terminal heterogeneity of the mtDNA is completely accounted for by the variability in the number of repeats. The 53 bp repeat does not resemble known telomeric DNA in sequence, repeat size, or number of repetitions. The termini occur at heterogeneous positions within the 53 bp repeat. The junction of the telomeric repeat with the internal DNA is at a different position within the telomeric repeat on each end of the mtDNA. We propose a model for the maintenance of the mtDNA ends involving unequal homologous recombination.     

Linear mitochondrial DNAs from yeasts: telomeres with large tandem repetitions

The terminal structure of the linear mitochondrial DNA (mtDNA) from the yeast Candida parapsilosis was investigated. This mtDNA, 30 kb long, has symmetrical ends forming inverted terminal repeats. These repeats are made up of a variable number of tandemly repeating units of 738 by each; the terminal nucleotide corresponds to a precise position within the last repeat unit sequence. The ends had an open structure accessible to enzymes, with a 5 single-stranded extension of about 110 nucleotides. No circular forms were detected in the DNA preparations. Two other unrelated species, Pichia philodendra and Candida salmanticensis also appear to have a linear mtDNA of similar organization. These linear DNAs (which we name Type 2 linear mtDNAs) are distinct from the previously described linear mtDNAs of yeasts whose termini are formed by a closed hairpin loop (Type 1 linear mtDNA). The terminal structure of C. parapsilosis mtDNA is reminiscent of the linear mitochondrial genomes of the ciliate Tetrahymena although, in the latter, the telomeric tandem repeat unit is considerably shorter.     

Telomeric repeats of Tetrahymena malaccensis mitochondrial DNA: a multimodal distribution that fluctuates erratically during growth.

The linear mitochondrial DNA (mtDNA) of Tetrahymena malaccensis has tandem 52-base-pair repeats at its telomeres. The mtDNA has a multimodal distribution of telomeres. Different groups in the distribution have different numbers of telomeric repeats. The standard deviation of the size of each end group is independent of the mean size of the end group. The two sides of the mtDNA have different multimodal distributions of repeats. Cloned cell lines have multimodal distributions of mtDNA telomeres distinct from that of the original cell line. The number of telomere end groups and the average size of the end groups change in an erratic fashion as the cells are passaged and do not reach a stable equilibrium distribution in 185 generations. We propose that the mean size of a telomere end group and the size distribution of an end group are independently regulated. The system controlling the average size of end groups may be defective in T. malaccensis, since a closely related species (T. thermophila) does not have a multimodal distribution of mtDNA telomeres. T. hyperangularis, which has different telomeric repeats on each side of its mtDNA, has a multimodal distribution of mtDNA telomeres on only one side, suggesting that the mechanism controlling the average number of repeats in an end group can be sequence specific. These mitochondrial telomeres provide a new example of the more general phenomenon of expansion and contraction of arrays of repeated sequences seen, for example, with simple-sequence "satellite" DNAs; however, the mitochondrial telomeres change on a very short time scale.     

Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA.

The sequences of the displacement-loop (D-loop) regions of mitochondrial DNA (mtDNA) from mouse L cells and human KB cells have been determined and provide physical maps to aid in the identification of sequences involved in the regulation of replication and expression of mammalian mtDNA. Both D-loop regions are bounded by the genes for tRNAPhe and tRNAPro. This region contains the most highly divergent sequences in mtDNA with the exceptions of three small conserved sequence blocks near the 5' ends of D-loop strands, a 225 nucleotide conserved sequence block in the center of the D-loop strand template region, and a short sequence associated with the 3' ends of D-loop strands. A sequence similar to that associated with the 3' termini of D-loop strands overlaps one of the conserved sequence blocks near the 5' ends of D-loop strands. The large, central conserved sequence probably does not code for a protein since no open reading frames are discretely conserved. Numerous symmetric sequences and potential secondary structures exist in these sequences, but none appear to be clearly conserved between species.     

Mitochondrial genome variability within the Candida parapsilosis species complex

Candida parapsilosis species complex includes three closely related species, namely C. parapsilosis (sensu stricto), C. orthopsilosis, and C. metapsilosis. Unlike most other yeast lineages, members of this species complex possess a linear mitochondrial genome. Yet, its circularized mutant form was identified in strains of C. orthopsilosis and C. metapsilosis. To investigate the underlying variability, we performed comparative analyses of the complete mitochondrial DNA sequences in a collection of strains. Our results demonstrate that in contrast to C. parapsilosis and C. metapsilosis, C. orthopsilosis exhibits remarkably high nucleotide diversity whose pattern is consistent with intraspecific genetic exchange.     

Mitochondrial DNA recombination in Brassica campestris

The structure of the mitochondrial genome in plants is unclear, but appears to consist of mostly linear DNA with some other structures, including branched molecules and subgenomic circles. Mitochondrial DNA (mtDNA) recombination was analyzed in Brassica campestris, which has one of the smallest mitochondrial genomes (218 kb) in higher plants. Field-inversion gel electrophoresis (FIGE) separated mtDNA into discrete populations that each represents the entire genome. Electron microscopy revealed large, mostly linear molecules trapped in the wells, slower migrating populations with mostly linear DNA and a low level of circular and networked mtDNA molecules of 10–140 kbp, and a fast migrating population of 10–50 kbp linear mtDNA. Some smaller than genome size circular molecules and circles with tails were observed, and may represent recombination or rolling circle replication intermediates. Hybridization of end-labeled mtDNA suggests there may be specific ends (or recombination hotspots) for some linear molecules. Analysis of mtDNA enriched by BND-cellulose and separated by two-dimensional agarose gel electrophoresis shows the presence of complex recombination structures and the presence of significant single-stranded regions in mtDNA. These findings provide further evidence that DNA recombination contributes to the complex structure of mtDNA in plants.     

Mitochondrial telomeres: surprising diversity of repeated telomeric DNA sequences among six species of Tetrahymena

The DNA sequences at the ends of the linear mtDNA of 6 species of Tetrahymena encompassing 13 strains were determined. All the strains have variable numbers of a tandemly repeated DNA sequence, 31 bp to 53 bp in size, at their mtDNA termini. Based upon the size and nucleotide sequence of the terminal repeats, the telomeres can be separated into four classes. T. pigmentosa, hyperangularis, and hegewischi have different telomeric repeats on the two ends of their mtDNAs. The only conserved feature of the mtDNA termini is the presence of tandem repeats. The function of the repeats might be to promote unequal crossing over during recombination, thereby overcoming the problem of telomere replication for these linear DNAs.     

Comparison of codon usage and tRNAs in mitochondrial genomes of Candida species

To gain insight into the nature of the mitochondrial genomes (mtDNA) of different Candida species, the synonymous codon usage bias of mitochondrial protein coding genes and the tRNAs in C. albicans, C. parapsilosis, C. stellata, C. glabrata and the closely related yeast Saccharomyces cerevisiae were analyzed. Common features of the mtDNA in Candida species are a strong A+T pressure on protein coding genes, and insufficient mitochondrial tRNA species are encoded to perform protein synthesis. The wobble site of the anticodon is always U for the NNR (NNA and NNG) codon families, which are dominated by A-ending codons, and always G for the NNY (NNC and NNU) codon families, which is dominated by U-ending codons, and always U for the NNN (NNA, NNU, NNC and NNG) codon families, which are dominated by A-ending codons and U-ending codons. Patterns of synonymous codon usage of Candida species can be classified into three groups: (1) optimal codon-anticodon usage, Glu, Lys, Leu (translated by anti-codon UAA), Gln, Arg (translated by anti-codon UCU) and Trp are containing NNR codons. NNA, whose corresponding tRNA is encoded in the mtDNA, is used preferentially. (2) Non-optimal codon-anticodon usage, Cys, Asp, Phe, His, Asn, Ser (translated by anti-codon GCU) and Tyr are containing NNY codons. The NNU codon, whose corresponding tRNA is not encoded in the mtDNA, is used preferentially. (3) Combined codon-anticodon usage, Ala, Gly, Leu (translated by anti-codon UAG), Pro, Ser (translated by anti-codon UGA), Thr and Val are containing NNN codons. NNA (tRNA encoded in the mtDNA) and NNU (tRNA not encoded in the mtDNA) are used preferentially. In conclusion, we propose that in Candida species, codons containing A or U at third position are used preferentially, regardless of whether corresponding tRNAs are encoded in the mtDNA. These results might be useful in understanding the common features of the mtDNA in Candida species and patterns of synonymous codon usage.     

Characterization of mitochondrial DNA in various Candida species: isolation, restriction endonuclease analysis, size, and base composition.

A practical and effective method for the extraction of mitochondrial DNA from Candida species was developed. Zymolyase was used to induce yeast protoplasts, and mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations. Restriction endonuclease analyses of mitochondrial DNAs from 19 isolates representing seven species of Candida (C. albicans, C. kefyr, C. lusitaniae, C. maltosa, C. parapsilosis, C. shehatae, and C. tropicalis) and Lodderomyces elongisporus revealed different cleavage patterns that appeared to be specific for the species. Few common restriction fragments were evident. The genome sizes of the mitochondrial DNAs ranged from 26.4 to 51.4 kilobase pairs, and the guanine-plus-cytosine contents ranged from 20.7 to 36.8 mol%. There was no correlation between the base compositions of nuclear and mitochondrial DNAs. Eight isolates of C. parapsilosis, including the type culture, and an ascosporogenous strain of L. elongisporus, which was once proposed as the teleomorph of C. parapsilosis, had similar mitochondrial DNA molecular sizes (30.2 and 28.8 kilobase pairs); however, restriction endonuclease patterns of these organisms were distinct. These data provide additional support for discrimination of these two species. The results of our experiments demonstrate that mitochondrial DNA analyses may provide useful criteria for the differentiation of yeast species.     

The structures at the termini of chromosomes: what is there hidden under the cap ?

The telomeres are the nucleoproteic structures present at the ends of eukaryotic chromosomes. One can compare them to the protective ends of a shoelace; when the ends get eroded, the shoelace disintegrates and we dispose of it. The same thus applies to the chromosomes; when telomeres reach a critical threshold for function, the genome becomes unstable and the cell senesces. Therefore, telomeres, and particularly their terminal DNA structures, are critical for the integrity of the genome.     

Accelerated aging as evidenced by increased telomere shortening and mitochondrial DNA depletion in patients with type 2 diabetes

Although shortened telomeres were shown associated with several risk factors of diabetes, there is lack of data on their relationship with mitochondrial dysfunction. Therefore, we compared the relationship between telomere length and mitochondrial DNA (mtDNA) content in patients with type 2 diabetes mellitus (T2DM; n = 145) and in subjects with normal glucose tolerance (NGT; n = 145). Subjects were randomly recruited from the Chennai Urban Rural Epidemiology Study. mtDNA content and telomere length were assessed by Real-Time PCR. Malonodialdehyde, a marker of lipid peroxidation was measured by thiobarbituric acid reactive substances (TBARS) using fluorescence methodology. Adiponectin levels were measured by radioimmunoassay. Oxidative stress as determined by lipid peroxidation (TBARS) was significantly (p < 0.001) higher in patients with T2DM compared to NGT subjects. In contrast, the mean telomere length, adiponectin and mtDNA content were significantly (p < 0.001) lower in patients with T2DM compared to NGT subjects. Telomere length was positively correlated with adiponectin, HDL, mtDNA content and good glycemic/lipid control and negatively correlated with adiposity and insulin resistance. On regression analysis, shortened telomeres showed significant association with T2DM even after adjusting for waist circumference, insulin resistance, triglyceride, HDL, adiponectin, mtDNA & TBARS. mtDNA depletion showed significant association with T2DM after adjusting for waist circumference and adiponectin but lost its significance when further adjusted for telomere length, TBARS and insulin resistance. Our study emphasizes the clustering of accelerated aging features viz., shortened telomeres, decreased mtDNA content, hypoadiponectinemia, low HDL, and increased oxidative stress in Asian Indian type 2 diabetes patients.     

DNA damage in telomeres and mitochondria during cellular senescence: is there a connection?

Cellular senescence is the ultimate and irreversible loss of replicative capacity occurring in primary somatic cell culture. It is triggered as a stereotypic response to unrepaired nuclear DNA damage or to uncapped telomeres. In addition to a direct role of nuclear DNA double-strand breaks as inducer of a DNA damage response, two more subtle types of DNA damage induced by physiological levels of reactive oxygen species (ROS) can have a significant impact on cellular senescence: Firstly, it has been established that telomere shortening, which is the major contributor to telomere uncapping, is stress dependent and largely caused by a telomere-specific DNA single-strand break repair inefficiency. Secondly, mitochondrial DNA (mtDNA) damage is closely interrelated with mitochondrial ROS production, and this might also play a causal role for cellular senescence. Improvement of mitochondrial function results in less telomeric damage and slower telomere shortening, while telomere-dependent growth arrest is associated with increased mitochondrial dysfunction. Moreover, telomerase, the enzyme complex that is known to re-elongate shortened telomeres, also appears to have functions independent of telomeres that protect against oxidative stress. Together, these data suggest a self-amplifying cycle between mitochondrial and telomeric DNA damage during cellular senescence.     

Association of G-quadruplex forming sequences with human mtDNA deletion breakpoints

Background

Mitochondrial DNA (mtDNA) deletions cause disease and accumulate during aging, yet our understanding of the molecular mechanisms underlying their formation remains rudimentary. Guanine-quadruplex (GQ) DNA structures are associated with nuclear DNA instability in cancer; recent evidence indicates they can also form in mitochondrial nucleic acids, suggesting that these non-B DNA structures could be associated with mtDNA deletions. Currently, the multiple types of GQ sequences and their association with human mtDNA stability are unknown.

Results

Here, we show an association between human mtDNA deletion breakpoint locations (sites where DNA ends rejoin after deletion of a section) and sequences with G-quadruplex forming potential (QFP), and establish the ability of selected sequences to form GQ in vitro. QFP contain four runs of either two or three consecutive guanines (2G and 3G, respectively), and we identified four types of QFP for subsequent analysis: intrastrand 2G, intrastrand 3G, duplex derived interstrand (ddi) 2G, and ddi 3G QFP sequences. We analyzed the position of each motif set relative to either 5'' or 3'' unique mtDNA deletion breakpoints, and found that intrastrand QFP sequences, but not ddi QFP sequences, showed significant association with mtDNA deletion breakpoint locations. Moreover, a large proportion of these QFP sequences occur at smaller distances to breakpoints relative to distribution-matched controls. The positive association of 2G QFP sequences persisted when breakpoints were divided into clinical subgroups. We tested in vitro GQ formation of representative mtDNA sequences containing these 2G QFP sequences and detected robust GQ structures by UV–VIS and CD spectroscopy. Notably, the most frequent deletion breakpoints, including those of the "common deletion", are bounded by 2G QFP sequence motifs.

Conclusions

The potential for GQ to influence mitochondrial genome stability supports a high-priority investigation of these structures and their regulation in normal and pathological mitochondrial biology. These findings emphasize the potential importance of helicases that subsequently resolve GQ to maintain the stability of the mitochondrial genome.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-677) contains supplementary material, which is available to authorized users.     

The complete mitochondrial genome of the yeast Pichia sorbitophila

Here, we report the complete nucleotide sequence of the 39 107-bp mitochondrial genome of the yeast Pichia sorbitophila . This genome is closely related to those of Candida parapsilosis and Debaryomyces hansenii , as judged from sequence similarities and synteny conservation. It encodes three subunits of cytochrome oxidase ( COX1, COX2 and COX3 ), three subunits of ATP synthase ( ATP6, ATP8 and ATP9 ), the seven subunits of NADH dehydrogenase ( NAD1-6 and NAD4L ), the apocytochrome b ( COB ), the large and small rRNAs and a complete set of tRNAs. Although the mitochondrial genome of P. sorbitophila contains the same core of mitochondrial genes observed in the ascomycetous yeasts, those coding for the RNAse P and the ribosomal protein VAR1p are missing. Moreover, the mtDNA of P. sorbitophila contains several introns in its genes and has the particularity of possessing an intron, which is not linked to any upstream exon.