Yeast as a model for human mtDNA replication.

The recognition, approximately a decade ago, that mutations in mtDNA can impair oxidative phosphorylation (OXPHOS) capacity and lead to human diseases (Holt et al. 1988; Wallace et al. 1988) heightened interest in the regulation and maintenance of this multicopy, extranuclear genome. Since then, mitochondrial dysfunction resulting from mtDNA mutation, instability, or copy-number deregulation has been implicated in numerous pathological conditions and the normal aging process (Wallace 1992; Larsson and Clayton 1995). The preservation of a functional mitochondrial genome over an individual's lifetime requires not only proper replication, segregation, and expression of functional genetic units during development and all subsequent mitotic cell divisions but also protection from and efficient repair of mtDNA damage. As I outline below, our current understanding of basic principles underlying many of these processes has come from studies of the budding yeast, Saccharomyces cerevisiae. Beyond the many fundamental similarities of mtDNA replication in human and yeast cells, however, there are aspects of the process that differ between our two species. These differences, which may, in some cases, limit the utility of S. cerevisiae as a model for human mtDNA replication, need to be considered in the discussion of the etiology of mitochondrial diseases. Human mtDNA has a number of characteristics that confound our understanding of mitochondrial genetics. First, multiple copies of mtDNA are present in most cell types and within each organelle. The copy number is tightly regulated and typically is 102–104 copies/cell, depending on cell type. Second, a mixture of wild-type and mutated mtDNA molecules can coexist in the same cell, tissue, or individual. At a cellular level, such a heteroplasmic state complicates the segregation pattern of mtDNA mutations, because an apparently random subset of the total mtDNA population is inherited by each daughter cell during cell division. Depending on the percentage of wild-type and mutated mtDNA molecules delivered to and propagated in each daughter cell, the degree of heteroplasmy can drift dramatically, even from undetectable levels of mutant mtDNA to predominantly mutant mtDNA, in the course of a single cell division. Because this phenomenon can also occur in the female germline during either mitotic or meiotic divisions, large shifts in the degree of heteroplasmy are also often observed between mothers and their children. Finally, phenotypic expression of mtDNA mutations exhibit a threshold effect, whereby the percentage of mutated mtDNA molecules in a cell or tissue must reach a critical high percentage (60%–90%) before a defect in OXPHOS is manifest. At percentages of mutant mtDNA that are below this threshold, mtDNA mutations appear to be phenotypically silent. These factors contribute to the complicated tissue-specific and variably penetrant phenotypic-expression patterns of mtDNA mutations observed in individuals affected by mitochondrial genetic disease (Grossman and Shoubridge 1996). Mitochondrial genomes from different species vary dramatically in size, structure, and coding capacity. In all cases, mtDNA encodes a small subset of the ∼80 protein subunits of the OXPHOS enzyme complexes in the mitochondrial inner membrane, which together generate cellular ATP. Human mtDNA is a 16.5-kb double-stranded circular molecule (fig. 1A) that encodes 13 OXPHOS-related mRNAs, as well as 22 tRNAs and 2 rRNAs required to translate these messages on ribosomes in the mitochondrial matrix. The genome is remarkably compact, with all 37 genes efficiently organized into two polycistronic transcription units. A unique feature of these primary transcripts is that each rRNA and most of the mRNAs are flanked by at least one tRNA. This gene arrangement leads to a mode of gene expression that requires a large number of RNA-processing events in order to liberate the mature mRNA, tRNA, and rRNA species from these complex precursor transcripts. As with gene expression, replication of human mtDNA depends on RNA transcript processing, because the mtDNA polymerase polγ requires specific RNA oligonucleotides to prime initiation. Other than two related ribonucleoproteins, RNase MRP and RNase P, the factors that mediate these processing events in human mitochondria have remained elusive, and the regulatory system that coordinates mtRNA processing with other intracellular and physiological processes remains obscure. Suitable model systems that faithfully reproduce mitochondrial disease phenotypes are needed to approach these regulatory issues. Because the basic machinery of mtDNA transcription and replication appears to be well conserved among diverse eukaryotes, the experimentally tractable yeast cell provides a valuable system in which to characterize key mitochondrial regulatory molecules. Figure 1 A, Human mtDNA, a 16.5-kb double-stranded circular molecule, with the two DNA strands designated as H or L strands, on the basis of their relative buoyant densities in denaturing cesium-chloride gradients. OH is located immediately downstream of the LSP, .