Investigations of the structure and dynamics of chromosomes present in the mitochondria of eukaryotic cells provide an opportunity to uncover principles of organellar inheritance. In particular, we investigate the role(s) of various molecular forms of mitochondrial genomes in organellar inheritance and analyze mitochondrial DNA replication intermediates and their segregation during division of the eukaryotic cell. Studies of mitochondrial chromosomes go beyond the field of mitochondrial genetics and genomics. As many organisms harbor linear DNA genomes in their mitochondria, our work focuses on elucidating the nature and evolutionary emergence of telomere maintenance pathways, which play a key role in the processes of cellular senescence and immortalization.
Linear mitochondrial genomes were found in organisms belonging to diverse phylogenetic taxa such as ciliates, apicomplexan protozoa, green algae, yeasts, oomycetes, slime molds as well as in metazoan species from the phylum Cnidaria. Several lines of evidence indicate that the linear form of a mitochondrial genome has neither an independent evolutionary origin nor a radically different lifestyle from its circular counterpart. Rather, the conversion from one form to another has occurred accidentally through a relatively simple mechanism during evolution. This means that a cell with a linearized mitochondrial genome may use a tinkering strategy to mobilize a preexisting set of proteins to deal with the end-replication problem within its organelle. Our studies revealed that the fortuitous evolutionary emergence of linear DNA genomes in mitochondria was accompanied by the generation of various types of terminal structures, adaptation of components of DNA replication machinery, and hence the application of differing strategies of telomere replication.
Results from our laboratory demonstrate that the mitochondria of the pathogenic yeast Candida parapsilosis and two closely related species, Candida metapsilosis and Candida orthopsilosis, contain compact linear DNA genomes terminating with specific telomeric structures. Similar to the termini of eukaryotic nuclear chromosomes, their mitochondrial counterparts consist of terminal arrays of tandem repeats and a single-stranded overhang protected by a specific mitochondrial telomere-binding protein. Moreover, the ends of linear mitochondrial DNA were shown to adopt a higher-order structure termed the telomeric loop (t-loop), which is analogous to the t-loops present at the tips of mammalian chromosomes. Such structural similarities indicate that mitochondrial telomeres play essentially the same biological roles as their nuclear equivalents—that is, they ensure the complete replication of the linear DNA molecules and mask the ends from DNA repair machinery as well as protect the ends from exonucleolytic degradation and end-to-end fusions. Analysis of the mitochondrial system revealed yet another general, evolutionarily conserved characteristic of telomeres. In addition to linear DNA, the mitochondria of C.parapsilosis, C. metapsilosis, C. orthopsilosis, Candida salmanticensis, and Pichia philodendri contain series of extragenomic circular molecules consisting exclusively of integral multimers of t-circles. Recently, we demonstrated that the t-circles replicate via a rolling circle strategy, which generates amplified linear arrays of the telomeric sequence. Moreover, we found that mutant strains lacking t-circles contain a circularized derivative of the genome formed by fusion of the left and right telomeres. Experiments with isogenic mutant strains differing in the form of mitochondrial genome revealed that, at least in certain circumstances, the genome linearity and/or the presence of telomeres may provide a growth advantage to the host.
The t-circle–dependent mechanism of telomere synthesis does not require the activity of telomerase and appears to represent the main, or perhaps the only, pathway of telomere maintenance operating in yeast mitochondria. Given that t-circles were identified in the nuclei of organisms ranging from yeasts to vertebrates, the mechanism may be of general significance. Importantly, recent studies from several laboratories demonstrate that t-circles represent a specific feature of some human ALT (alternative lengthening of telomeres) cell lines and, furthermore, may be implicated in both telomere maintenance and the sudden changes in telomere length known as telomere rapid deletion. Investigations of mitochondrial telomere dynamics thus may have implications for understanding the nature of telomerase-independent mechanisms of telomere maintenance operating in a significant number of human cancers. These studies may also clarify how the ALT pathways arise in telomerase-deficient or drug-treated cells.
We proposed that the emergence of linear DNA genomes in mitochondria provides an evolutionary paradigm for the origin of linear chromosomes and telomere replication pathways in the nuclei of early eukaryotes. The role of t-circles in telomere dynamics is in line with the hypothesis that ancestral eukaryotes used alternative mechanism(s) to maintain their nuclear chromosomes and that the telomerase was recruited later, providing the most robust way to preserve the chromosomal termini. Hence, the t-circles may represent molecular fossils of the primordial telomere maintenance system. Telomeric arrays produced with the assistance of t-circles might have subsequently mediated the formation of higher order structures, such as t-loops, at chromosomal termini, thus stabilizing the linear chromosomal form by fulfilling essential telomeric functions.
Because our main experimental model system is the pathogenic yeast C. parapsilosis, we continually develop tools for its genetic manipulation. We prepared suitable host strains and genomic DNA libraries and constructed a set of replicative plasmid vectors with selection and reporter markers for gene cloning and expression and the intracellular localization of protein products of cloned genes. We also optimized protocols for transferring DNA into yeast cells.
We are continuing our efforts to draw a complex portrait of mitochondrial chromosomes and to reconstruct their three-dimensional structure. Mitochondrial nucleoids are specific DNA protein complexes representing fundamental segregating units of the organellar genome. Identifying the roles of individual nucleoid components and elucidating how their functions are orchestrated to ensure the packaging, stability, and segregation of the mitochondrial DNA are crucial for understanding the molecular principles that govern mitochondrial inheritance and genome evolution. We systematically analyze protein components of the nucleoids and enzymes implicated in mitochondrial DNA replication from C. parapsilosis. Comparing these proteins with their homologues from other yeast species offers a unique insight into the evolution of the organellar chromosome and provides a basis for further functional studies. In particular, we are interested in how mitochondrial DNA molecules are packaged into compact nucleoid structures. Comparative analysis revealed that C. parapsilosis possesses essentially the same set of nucleoid proteins as other yeast species. However, the proteins known to directly interact with the mitochondrial DNA are the least conserved components of the nucleoid. Why those proteins evolve at a higher rate remains to be determined.
Last updated September 2008