Title: Yeast as a Model for Human mtDNA Replication
Abstract: The recognition, approximately a decade ago, that mutations in mtDNA can impair oxidative phosphorylation (OXPHOS) capacity and lead to human diseases (Holt et al. Holt et al., 1988Holt IJ Harding AE Morgan-Hughes JA Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies.Nature. 1988; 331: 717-719Crossref PubMed Scopus (1446) Google Scholar; Wallace et al. Wallace et al., 1988Wallace DC Singh G Lott MT Hodge JA Schurr TG Lezza AMS Elsas II, LJ et al.Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy.Science. 1988; 242: 1427-1430Crossref PubMed Scopus (1827) Google Scholar) 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 Wallace, 1992Wallace DC Diseases of mitochondrial DNA.Annu Rev Biochem. 1992; 61: 1175-1212Crossref PubMed Scopus (1158) Google Scholar; Larsson and Clayton Larsson and Clayton, 1995Larsson N Clayton DA Molecular genetic aspects of human mitochondrial disorders.Annu Rev Genet. 1995; 29: 151-178Crossref PubMed Scopus (390) Google Scholar). 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 Grossman and Shoubridge, 1996Grossman LI Shoubridge EA Mitochondrial genetics and human disease.Bioessays. 1996; 18: 983-991Crossref PubMed Scopus (91) Google Scholar). 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. In S. cerevisiae, as in all mitochondrial eukaryotes, a large number of biochemical reactions occur in mitochondria that make the organelles themselves indispensable for life. However, because yeast are facultative anaerobes, they are not entirely dependent on OXPHOS for the production of ATP and NAD+. In other words, yeast can survive without a functional mitochondrial respiratory chain, provided that an appropriate carbon source (e.g., glucose) is made available to permit continued ATP production via glycolysis and fermentation. Since the mitochondrial genetic system functions only to provide key OXPHOS enzyme subunits, yeast grown on fermentable carbon sources are viable even if mtDNA is mutated or absent. Respiration-deficient mutants of yeast, referred to as "petite" because of their slow growth and small colony size, can arise if either mtDNA or nuclear-DNA mutations compromise OXPHOS. The ability to analyze petite mutants has made yeast an attractive experimental system for isolation and characterization of nuclear-gene products required for mitochondrial function and biogenesis. Three examples of conservation between human and yeast mtDNA metabolism will suffice to demonstrate the utility of this system. First, the initial gene encoding a dedicated mtRNA polymerase (RPO41) was identified because mutations in this gene confer a petite phenotype, and this gene has been shown to be required not only for mitochondrial transcription but also for maintenance of mtDNA in cells (Greenleaf et al. Greenleaf et al., 1986Greenleaf AL Kelly JL Lehman IR Yeast RPO41 gene product is required for transcription and maintenance of the mitochondrial genome.Proc Natl Acad Sci USA. 1986; 83: 3391-3394Crossref PubMed Scopus (103) Google Scholar). Perhaps surprisingly, nucleotide-sequence analysis has revealed that yeast mtRNA polymerase is homologous to the simple RNA polymerases encoded by the Escherichia coli bacteriophage genomes of T7, T3, and SP6 (Masters et. al. Masters et al., 1987Masters BS Stohl LL Clayton DA Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7.Cell. 1987; 51: 89-99Abstract Full Text PDF PubMed Scopus (303) Google Scholar). It is now clear that most, if not all, eukaryotic mtRNA polymerases, including human (Tiranti et al. Tiranti et al., 1997Tiranti V Savoia A Forti F D'Apolito M Centra M Rocchi M Zeviani M Identification of the gene encoding the human mitochondrial RNA polymerase (hmtRPOL) by cyberscreening of the expressed sequence tags database.Hum Mol Genet. 1997; 6: 615-625Crossref PubMed Scopus (143) Google Scholar), conform to this bacteriophage model. Second, the first gene encoding an mtDNA polymerase was also discovered in yeast (MIP1) (Foury Foury, 1989Foury F Cloning and sequencing of the nuclear gene MIP1 encoding the catalytic subunit of the yeast mitochondrial DNA polymerase.J Biol Chem. 1989; 264: 20552-20560Abstract Full Text PDF PubMed Google Scholar), revealing that it is a member of the DNA Pol I family (family A) of DNA polymerases (Braithwaite and Ito Braithwaite and Ito, 1993Braithwaite D Ito J Compilation, alignment, and phylogenetic relationships of DNA polymerases.Nucleic Acids Res. 1993; 21: 787-802Crossref PubMed Scopus (521) Google Scholar). In addition, the MIP1 gene sequence revealed the presence of three exonuclease domains in the protein, providing the first compelling evidence that mtDNA polymerases do indeed have intrinsic proofreading capacity. Finally, genes encoding eukaryotic homologues of the bacterial DNA mismatch-repair protein MutS were identified initially in yeast. One of these proteins, Mshlp, is located in the mitochondrial matrix and is required for proper maintenance of mtDNA (Reenan and Kolodner Reenan and Kolodner, 1992Reenan RA Kolodner RD Characterization of insertion mutations in the Saccharomyces cerevisiae MSH1 and MSH2 genes: evidence for separate mitochondrial and nuclear functions.Genetics. 1992; 132: 975-985Crossref PubMed Google Scholar), which suggests that yeast mitochondria have a critical DNA mismatch-recognition and/or -repair capacity. Bogenhagen (Bogenhagen, 1999Bogenhagen DF Repair of mtDNA in vertebrates.Am J Hum Genet. 1999; 64: 1276-1281Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) recently has reviewed evidence for several forms of mtDNA repair that occur in mammalian cells as well. These examples highlight only a few of the advances in our understanding of mtDNA regulation that have resulted from the study of the yeast model system. For a more extensive discussion of the contribution of yeast studies to the understanding of mitochondrial-gene expression, mitochondrial inheritance, and nuclear-mitochondrial communication, see the work of Costanzo and Fox (Costanzo and Fox, 1990Costanzo MC Fox TD Control of mitochondrial gene expression in Saccharomyces cerevisiae.Annu Rev Genet. 1990; 24: 91-113Crossref PubMed Google Scholar), Shadel and Clayton (Shadel and Clayton, 1993Shadel GS Clayton DA Mitochondrial transcription initiation: variation and conservation.J Biol Chem. 1993; 268: 16083-16086Abstract Full Text PDF PubMed Google Scholar), Dieckmann and Staples (Dieckmann and Staples, 1994Dieckmann CL Staples RR Regulation of mitochondrial gene expression in Saccharomyces cerevisiae.Int Rev Cytol. 1994; 152: 145-181Crossref PubMed Scopus (74) Google Scholar), Grivell (Grivell, 1995Grivell L Nucleo-mitochondrial interactions in mitochondrial gene expression.Crit Rev Biochem Mol Biol. 1995; 30: 121-164Crossref PubMed Scopus (139) Google Scholar), Poyton and McEwan (Poyton and McEwen, 1996Poyton RO McEwen JE Crosstalk between nuclear and mitochondrial genomes.Annu Rev Biochem. 1996; 65: 563-607Crossref PubMed Scopus (414) Google Scholar), Hermann and Shaw (Hermann and Shaw, 1998Hermann GJ Shaw JM Mitochondrial dynamics in yeast.Annu Rev Cell Dev Biol. 1998; 14: 265-303Crossref PubMed Scopus (152) Google Scholar), and Yaffe (Yaffe, 1999Yaffe MP The machinery of mitochondrial inheritance and behavior.Science. 1999; 283: 1493-1497Crossref PubMed Scopus (412) Google Scholar). The mechanism of human mtDNA replication was determined in broad outline by analysis of replication intermediates isolated from cultured cells (reviewed by Clayton Clayton, 1982Clayton DA Replication of animal mitochondrial DNA.Cell. 1982; 28: 693-705Abstract Full Text PDF PubMed Scopus (880) Google Scholar). These early studies led to the discovery of a major mtDNA molecular form that contains a displacement-loop (D-loop), a stable three-stranded DNA structure containing a short nascent mtDNA strand at the origin of heavy (H)-strand replication (OH; see fig. 1). This D-loop itself occurs within a larger noncoding region of the mtDNA molecule (referred to as the "D-loop regulatory region") that harbors not only OH but also the mitochondrial transcription promoters (fig. 1), making it the main control site for mtDNA replication and transcription. Vertebrate mtDNA also has a second origin of replication for the light (L)-strand (OL). OH and OL are unidirectional origins (i.e., DNA synthesis proceeds only in one direction) and are physically separated from each other on the mtDNA molecule (fig. 1). Thus, human mtDNA replicates by an unusual asynchronous mechanism that is initiated at OH (Clayton Clayton, 1982Clayton DA Replication of animal mitochondrial DNA.Cell. 1982; 28: 693-705Abstract Full Text PDF PubMed Scopus (880) Google Scholar). Because the entire mtDNA replication process is initiated at OH, events that lead to priming of H-strand replication in the D-loop region have been and continue to be a focus of intensive investigation. Initial insight into the mechanism of H-strand replication priming came with the determination that the mitochondrial L-strand and H-strand promoters (LSP and HSP, respectively) are located immediately adjacent to OH, suggesting that transcription and mtDNA replication might be linked. Indeed, detailed mapping and molecular characterization of D-loop RNA and DNA species revealed that RNA transcripts initiated at the LSP serve to prime H-strand DNA synthesis (Clayton Clayton, 1991Clayton DA Replication and transcription of vertebrate mitochondrial DNA.Annu Rev Cell Biol. 1991; 7: 453-478Crossref PubMed Scopus (511) Google Scholar). Considerable progress has been made in the understanding of this transcription-dependent mtDNA-replication mechanism, including the isolation and characterization of key, nucleus-encoded factors that interact at the D-loop region to promote transcription and replication of the mitochondrial genome. These data support the general model for initiation of H-strand mtDNA replication in humans that has been described by Shadel and Clayton (Shadel and Clayton, 1997Shadel GS Clayton DA Mitochondrial DNA maintenance in vertebrates.Annu Rev Biochem. 1997; 66: 409-435Crossref PubMed Scopus (770) Google Scholar). In this model, an RNA transcript is initiated at the LSP by human mtRNA polymerase. This event requires the nucleus-encoded transcription factor h-mtTFA (Parisi and Clayton Parisi and Clayton, 1991Parisi MA Clayton DA Similarity of human mitochondrial transcription factor 1 to high mobility group proteins.Science. 1991; 252: 965-969Crossref PubMed Scopus (426) Google Scholar), which binds to the region immediately upstream of the LSP and activates specific transcription initiation by human mtRNA polymerase. Next, as mtRNA polymerase traverses a series of three conserved-sequence blocks (CSB I–CSB III) at OH, the elongating LSP transcript forms an RNA/DNA hybrid that serves as a substrate for processing reactions that generate mature RNA primers (fig. 1). On the basis of in vitro analysis (Lee and Clayton Lee and Clayton, 1998Lee DY Clayton DA Initiation of mitochondrial DNA replication by transcription and R-loop processing.J Biol Chem. 1998; 273: 30614-30621Crossref PubMed Scopus (116) Google Scholar) and subcellular-localization studies (Li et al. Li et al., 1994Li K Smagula CS Parsons WJ Richardson JA Gonzalez M Hagler HK Williams RS Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis.J Cell Biol. 1994; 124: 871-882Crossref PubMed Scopus (103) Google Scholar), the endoribonuclease RNase MRP appears to be a key player in the processing of a complex precursor RNA/DNA hybrid into mature RNA primers. These primers are recognized by DNA polγ and accessory proteins to initiate H-strand DNA synthesis, thus beginning a productive mtDNA-replication event. Although analysis of mtDNA from other vertebrates has revealed some species-specific sequence variability, the D-loop–region configurations suggest that, in all cases, the transcription-primed mechanism is conserved (Shadel and Clayton Shadel and Clayton, 1997Shadel GS Clayton DA Mitochondrial DNA maintenance in vertebrates.Annu Rev Biochem. 1997; 66: 409-435Crossref PubMed Scopus (770) Google Scholar). The degree of conservation of mtDNA replication in lower eukaryotes has been addressed most extensively in S. cerevisiae, and substantial evidence indicates that transcription priming also operates in yeast (fig. 1). Nonetheless, such models remain controversial because of significant differences that exist between human and yeast mitochondrial genetic systems. For example, yeast mtDNA is larger (∼80 kb) than human (16.5 kb) and, instead of two unidirectional origins, contains four putative origins of replication (termed "ori/rep" sequences) that are bidirectional in nature. The greater complexity of yeast mtDNA replication has slowed progress in this system, but this disadvantage may be offset by the existence of a remarkable class of spontaneous mtDNA deletion mutants, the ρ− petites, which are easily identified and studied in yeast. These mutant strains often retain multiple copies of particular ori/rep sequences in mitochondria and have been used extensively to address mtDNA-replication and -segregation issues in yeast. Unfortunately, it remains unclear to what extent the behavior of these repetitive mutated mtDNAs reflects that of the wild-type mitochondrial genome. As discussed below, these issues not only complicate the analysis of wild-type mtDNA replication in yeast but also may limit to some degree the applicability of yeast as a model system for the understanding of human mitochondrial genetic phenomena. Under normal growth conditions, all laboratory yeast strains spontaneously generate petite mutants at a frequency of ∼1%/generation, largely because yeast mtDNA is prone to massive deletion and rearrangement. The mtDNA sequences retained in the resulting ρ− petite mutants often consist of amplified head-to-tail repeats of a portion of the genome, almost always containing <1% of the normal sequence. These repeat genomes may segregate in unusual ways. For example, when the so-called hypersuppressive (HS) ρ− mutants are mated to a strain harboring a wild-type mitochondrial genome, the HS ρ− genome is inherited preferentially to the wild-type genome by the daughter cells emerging from the cross. In the vast majority of HS mutants, the repeated region includes an ori/rep sequence (Blanc and Dujon Blanc and Dujon, 1980Blanc H Dujon B Replicator regions of the yeast mitochondrial DNA responsible for suppressiveness.Proc Natl Acad Sci USA. 1980; 77: 3942-3946Crossref PubMed Scopus (97) Google Scholar; de Zamaroczy et al. de Zamaroczy et al., 1981de Zamaroczy M Marotta R Faugeron-Fonty G Goursot R Mangin M Baldacci G Bernardi G The origins of replication of the yeast mitochondrial genome and the phenomenon of suppressivity.Nature. 1981; 292: 75-78Crossref PubMed Scopus (101) Google Scholar), an ∼280 bp sequence that is structurally similar to the vertebrate OH. Wild-type yeast mitochondrial genomes contain as many as eight such sequences, of which four (ori1–ori3 and ori5) harbor an intact transcriptional promoter and likely represent bona fide origins of DNA replication. In studies of various HS strains, Baldacci et al. (Baldacci et al., 1984Baldacci G Chérif-Zahar B Bernardi G The initiation of DNA replication in the mitochondrial genome of yeast.EMBO J. 1984; 3: 2115-2120PubMed Google Scholar) found that RNA transcripts initiated at the ori/rep promoters are used as primers for mtDNA synthesis; more-recent work has shown that the essential oligoribonucleotide primers are 9–17 nucleotides long and hybridize at various sites encompassing the conserved CSB II element (Graves et al. Graves et al., 1998Graves T Dante M Eisenhour L Christianson TW Precise mapping and characterization of the RNA primers of DNA replication for a yeast hypersuppressive petite by in vitro capping with guanylyltransferase.Nucleic Acids Res. 1998; 26: 1309-1316Crossref PubMed Scopus (21) Google Scholar). In at least two of the four ori/rep sequences, CSB II is also the site at which a transcription-dependent RNA/DNA hybrid forms in vitro, suggesting that a longer RNA species that is transcribed in this region serves as the precursor of the oligoribonucleotide primers that are used in replication (Xu and Clayton Xu and Clayton, 1995Xu B Clayton DA A persistent RNA-DNA hybrid is formed during transcription at a phylogenetically conserved mitochondrial DNA sequence.Mol Cell Biol. 1995; 15: 580-589Crossref PubMed Scopus (103) Google Scholar). Furthermore, cells lacking either mtRNA polymerase or the mitochondrial transcription-initiation factor sc-mtTFB generate ρ− and ρ0 daughters (the latter of which entirely lacks mtDNA) at an increased rate (Greenleaf et al. Greenleaf et al., 1986Greenleaf AL Kelly JL Lehman IR Yeast RPO41 gene product is required for transcription and maintenance of the mitochondrial genome.Proc Natl Acad Sci USA. 1986; 83: 3391-3394Crossref PubMed Scopus (103) Google Scholar; Lisowsky and Michaelis Lisowsky and Michaelis, 1988Lisowsky T Michaelis G A nuclear gene essential for mitochondrial replication suppresses a defect of mitochondrial transcription in Saccharomyces cerevisiae.Mol Gen Genet. 1988; 214: 218-223Crossref PubMed Scopus (75) Google Scholar; Wang and Shadel Wang and Shadel, 1999Wang Y Shadel GS Stability of the mitochondrial genome requires an amino-terminal domain of yeast mitochondrial RNA polymerase.Proc Natl Acad Sci USA. 1999; 96: 8046-8051Crossref PubMed Scopus (53) Google Scholar). All of these data are consistent with an ori/rep-dependent, transcription-primed replication mechanism that is required for leading-strand synthesis of yeast mtDNA, similar to the mechanism proposed for vertebrate OH (fig. 1). Nonetheless, several features of yeast mtDNA both complicate the interpretation of the data that lead to this model and make wild-type mtDNA replication difficult to address experimentally. Despite the wealth of information that HS petite mutants have provided regarding mtDNA replication and segregation, complications persist about the proper analysis of these mutants. For instance, Fangman et al. (Fangman et al., 1989Fangman WL Henly JW Churchill G Brewer BJ Stable maintenance of a 35-base-pair yeast mitochondrial genome.Mol Cell Biol. 1989; 9: 1917-1921Crossref PubMed Scopus (54) Google Scholar) identified stable ρ− genomes that do not contain ori/rep sequences, and they later demonstrated that certain ori/rep-containing HS genomes can be maintained in the absence of mtRNA polymerase (Fangman et al. Fangman et al., 1990Fangman WL Henly JW Brewer BJ RPO41-independent maintenance of [ρ−] mitochondrial DNA in Saccharomyces cerevisiae.Mol Cell Biol. 1990; 10: 10-15PubMed Google Scholar). Furthermore, the involvement of recombination intermediates in the preferential inheritance patterns observed for HS genomes (i.e., the hypersuppressive phenomenon) also has been documented (Lockshon et al. Lockshon et al., 1995Lockshon D Zweifel SG Freeman-Cook LL Lorimer HE Brewer BJ Fangman WL A role for recombination junctions in the segregation of mitochondrial DNA in yeast.Cell. 1995; 81: 947-955Abstract Full Text PDF PubMed Scopus (141) Google Scholar). Altogether, these observations have led to the realization that HS genomes can propagate by mechanisms that are independent of ori/rep sequences and mtRNA polymerase-driven transcription. In theory, the highly repetitive nature of ρ− genomes would increase the potential for genetic recombination within and between these mtDNA molecules. Thus, a mode of replication for HS genomes that is dependent on the formation of recombination intermediates is one formal possibility that has been proposed (MacAlpine et al. MacAlpine et al., 1998MacAlpine DM Perlman PS Butow RA The high-mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo.Proc Natl Acad Sci USA. 1998; 95: 6739-6743Crossref PubMed Scopus (93) Google Scholar, and references within). In this regard, it should be stressed that aggressive genetic recombination, as is observed in yeast mitochondria, is not found in human mitochondria. Despite the identification of human mitochondrial activities potentially involved in recombination, homologous recombination between mtDNA molecules has yet to be demonstrated unambiguously in human cells (reviewed by Howell Howell, 1997Howell N mtDNA recombination: what do in vitro data mean?.Am J Hum Genet. 1997; 61: 19-22Abstract Full Text PDF PubMed Scopus (27) Google Scholar). A mode of replication that involves recombination intermediates, such as that proposed for yeast HS genomes, is therefore almost certainly not involved in the replication of vertebrate mtDNA, highlighting yet another salient difference between yeast and human mtDNA metabolism. The relevance of the above-mentioned properties of HS genomes to the mode of replication of wild-type yeast mtDNA also remains speculative. The existence of an alternative mode of replication for ρ− mtDNA need not argue against the proposed transcription-dependent (and ori/rep sequence-dependent) mode of replication for wild-type genomes. Likewise, the presence of a primarily transcription-dependent replication mechanism for wild-type mtDNA does not rule out the possibility that other modes of mtDNA replication may operate under certain circumstances. For example, a decrease or loss of mitochondrial transcription could result in a switch from a transcription-dependent to a transcription-independent (e.g., recombination-mediated) mode of mtDNA replication. Indeed, the complex assortment of mtDNA 5′ and 3′ ends and double-strand breaks at yeast ori1 in vivo may indicate that these sequences are recombinogenic (Van Dyck and Clayton Van Dyck and Clayton, 1998Van Dyck E Clayton DA Transcription-dependent DNA transactions in the mitochondrial genome of a yeast hypersuppressive petite mutant.Mol Cell Biol. 1998; 18: 2976-2985PubMed Google Scholar). Further speculation suggests that it is possible that the inherent instability of wild-type yeast mtDNA under normal growth conditions is due in part to the simultaneous occurrence of replication and recombination at ori/rep sequences, which might be expected to promote deletions or rearrangements. Perhaps even more surprising than the normal high level of mtDNA instability is the number of nuclear-gene mutations in yeast that enhance this instability (table 1). Many of these loci can be rationalized easily, especially in light of an accepted model of transcription-coupled replication. These include MIP1, which encodes DNA polγ; MTF1, which encodes the transcription-initiation factor sc-mtTFB; and RIM1, which encodes a mitochondrial single-strand DNA-binding protein (mtSSB). Other loci have little obvious connection to mtDNA metabolism and cannot be explained as easily. For example, mutations in genes for a Lon protease homologue (PIM1); an enzyme involved in branched-chain amino acid synthesis (ILV5); and a putative mitochondrial solute carrier (RIM2/MRS12) all promote the formation of ρ− or ρ0 cells. Evidently, the mtDNA-instability phenotype in yeast can result from any number of nuclear- or mitochondrial-gene mutations and therefore must be interpreted with some caution, particularly when studies in yeast are used to ascribe mtDNA-related functions to yeast homologues of human disease genes. For example, disruption of the yeast homologue (YFH1) of the human FRATAXIN gene has been shown to cause mtDNA instability in yeast, a phenotype that has not yet been reported in cells from individuals with Friedreich ataxia (reviewed in Knight et al. Knight et al., 1999Knight SAB Kim R Pain D Dancis A The yeast connection to Friedreich ataxia.Am J Hum Genet. 1999; 64: 365-371Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar).Table 1Yeast Genes Implicated in mtDNA StabilityGeneProtein Information and Mutant PhenotypeABF2HMG-box protein sc-mtTFA/mitochondrial nucleoid componentADR1Nuclear transcription factorERV1Null mutation lethal, affects mitochondrial transcript levelsFZO1Putative GTPase involved in mitochondrial membrane fusionILV5Enzyme in branched-chain amino acid biosynthesisMDM10Mitochondrial outer-membrane protein involved in mitochondrial inheritanceMDM12Mitochondrial outer-membrane protein involved in mitochondrial inheritanceMGM1Dynamin-like GTPase in outer mitochondrial membraneMGM101Mitochondrial nucleoid-component/oxidative mtDNA-damage responseMGM104Function unknownMHR1Implicated in mtDNA recombination and/or repairMIP1DNA polγMMM1Mitochondrial outer-membrane protein involved in mitochondrial inheritanceMSH1Bacterial MutS homologue located in mitochondriaMTF1sc-mtTFBMTF2/NAM1Putative RNA-processing and/or -transcription factorOCT1Yeast intermediate peptidase/protein import and iron homeostasisPET18Function unknown, PET18 mutation affecting multiple genesPET56Mitochondrial 21S rRNA methyltransferasePET112Posttranscriptional regulator of mitochondrial COX II genePET127Mitochondrial membrane-bound, putative RNA-processing factorPIF1mtDNA helicasePIM1Bacterial Lon protease homologue located in mitochondriaPPA2Mitochondrial inorganic pyrophosphataseRIM1mtSSBRIM2/MRS12Mitochondrial carrier-family homologueRPO41Core mtRNA polymeraseTTS1Cytoplasmic tyrosyl-tRNA synthetase, suppressing MGM104-1 mutan