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Mutations in mitochondrial DNA (mtDNA) are an important cause of disease and perhaps aging in human. DNA polymerase gamma (pol γ), the unique replicase inside mitochondria, plays a key role in the fidelity of mtDNA replication through selection of the correct nucleotide and 3'-5' exonuclease proofreading. For the first time, we have isolated and characterized antimutator alleles in the yeast pol γ (Mip1). These mip1 mutations, localised in the 3'-5' exonuclease and polymerase domains, elicit a 2-15 fold decrease in the frequency of mtDNA point mutations in an msh1-1 strain which is partially deficient in mtDNA mismatch-repair. In vitro experiments show that in all mutants the balance between DNA synthesis and exonucleolysis is shifted towards excision when compared to wild-type, suggesting that in vivo more opportunity is given to the editing function for removing the replicative errors. This results in partial compensation for the mismatch-repair defects and a decrease in mtDNA point mutation rate. However, in all mutants but one the antimutator trait is lost in the wild-type MSH1 background. Accordingly, the polymerases of selected mutants show reduced oligonucleotide primed M13 ssDNA synthesis and to a lesser extent DNA binding affinity, suggesting that in mismatch-repair proficient cells efficient DNA synthesis is required to reach optimal accuracy. In contrast, the Mip1-A256T polymerase, which displays wild-type like DNA synthesis activity, increases mtDNA replication fidelity in both MSH1 and msh1-1 backgrounds. Altogether, our data show that accuracy of wild-type Mip1 is probably not optimal and can be improved by specific (often conservative) amino acid substitutions that define a pol γ area including a loop of the palm subdomain, two residues near the ExoII motif and an exonuclease helix-coil-helix module in close vicinity to the polymerase domain. These elements modulate in a subtle manner the balance between DNA polymerization and excision.

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Mutations in DNA polymerase gamma (pol g), the unique replicase inside mitochondria, cause a broad and complex spectrum of diseases in human. We have used Mip1, the yeast pol g, as a model enzyme to characterize six pathogenic pol g mutations. Four mutations clustered in a highly conserved 3'-5' exonuclease module are localized in the DNA-binding channel in close vicinity to the polymerase domain. They result in an increased frequency of point mutations and high instability of the mitochondrial DNA (mtDNA) in yeast cells, and unexpectedly for mutator mutations in the exonuclease domain, they favour exonucleolysis versus polymerization. This trait is associated with highly decreased DNA-binding affinity and poorly processive DNA synthesis. Our data show for the first time that a 3'-5' exonuclease module of pol g plays a crucial role in the coordination of the polymerase and exonuclease functions and they strongly suggest that in patients the disease is not caused by defective proofreading but results from poor mtDNA replication generated by a severe imbalance between DNA synthesis and degradation.

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The yeast Saccharomyces cerevisiae has the capacity to survive large deletions or total loss of mtDNA (petite mutants), and thus in the last few years it has been used as a model system to study defects in mitochondrial DNA (mtDNA) maintenance produced by mutations in genes involved in mtDNA replication. In this paper we describe methods to obtain strains harboring mutations in nuclear genes essential for the integrity of mtDNA, to measure the frequency and the nature of petite mutants, to estimate the point mutation frequency in mtDNA and to determine whether a nuclear mutation is recessive or dominant and, in the latter case, the kind of dominance.

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Friedreich's ataxia is a neurodegenerative disease caused by the low expression of frataxin, a mitochondrial iron-binding protein which plays an important, but non-essential, role in the formation of iron-sulfur (Fe/S) clusters. It has been shown that Yfh1, the yeast frataxin homologue, interacts functionally and physically with Isu1, the scaffold protein on which the Fe/S clusters are assembled. The large beta-sheet platform of frataxin is a good ligand candidate for this interaction. We have generated 12 yeast mutants in conserved residues of the beta-sheet protruding at the surface or buried in the protein core. The Q129A, I130A, W131A(F) and R141A mutations, which reside in surface exposed residues of the fourth and fifth beta-strands, result in severe cell growth inhibition on high-iron media and low aconitase activity, indicating that Fe/S cluster biosynthesis is impaired. The null phenotype of the I130A mutant results from the high instability of the protein, pointing that this buried residue is essential for folding. In contrast, Gln-129, Trp-131 and Arg-141 residues which are spatially closely clustered define a patch important for protein function. Co-immunoprecipitation experiments using cell extracts show that W131A, unlike W131F, is the sole mutation that strongly decreases the interaction with Isu1. Therefore, Trp-131, which is the only strictly conserved frataxin residue in all sequenced species, appears as a major contributor to the interaction with Isu1 through its surface-exposed aromatic side chain.

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The yeast mitochondrial DNA (mtDNA) replicase Mip1 has been used as a model to generate five mutations equivalent to POLG mutations associated with a broad spectrum of diseases in human. All mip1 mutations, alone or in combination in cis or in trans, increase mtDNA instability as measured by petite frequency and Ery(R) mutant accumulation. This phenotype is associated with decreased Mip1 levels in mitochondrial extracts and/or decreased polymerase activity. We have demonstrated that (1) in the mip1(G651S) (hG848S) mutant the high mtDNA instability and increased frequency of point Ery(R) mutations is associated with low Mip1 levels and polymerase activity; (2) in the mip1(A692T-E900G) (hA889T-hE1143G) mutant, A692T is the major contributor to mtDNA instability by decreasing polymerase activity, and E900G acts synergistically by decreasing Mip1 levels; (3) in the mip1(H734Y)/mip1(G807R) (hH932Y/hG1051R) mutant, H734Y is the most deleterious mutation and acts synergistically with G807R as a result of its dominant character; (4) the mip1(E900G) (h1143G) mutation is not neutral but results in a temperature-sensitive phenotype associated with decreased Mip1 levels, a property explaining its synergistic effect with mutations impairing the polymerase activity. Thus, the human E1143G mutation is not a true polymorphism.

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In the Saccharomyces cerevisiae strains used for genome sequencing and functional analysis, the mitochondrial DNA replicase Mip1p contains a single nucleotide polymorphism changing the strictly conserved threonine 661 to alanine. This substitution is responsible for the increased rate of mitochondrial DNA point mutations and deletions in these strains.

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We describe methods that allow isolation, identification, and counting of mitochondrial mutants that are resistant to antibiotics (ant(R)) or respiratory deficient (rho-). (1) Analysis of diploid and meiotic progenies generated in crosses between mutants and tester strains allows distinguishing nuclear from mitochondrial mutants, for either antibiotic resistance or respiratory deficiency. (2) The mutation rate of mitochondrial deoxyribonucleic acid (mtDNA) can be estimated from the average frequency of ant(R) mutants produced in a large number of independent clones. (3) The frequency of retention of mtDNA fragments in rho- genomes accumulating in nuclear respiratory-deficient mutants can be determined by a genetic test based on the ability of these rho- genomes to restore cellular growth on glycerol in crosses with selected mutants bearing punctual mutations in their mtDNA (mit-).

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Friedreich ataxia is caused by decreased levels of frataxin, a mitochondrial acidic protein that is assumed to act as chaperone in the assembly of Fe-S clusters on the scaffold Isu protein. Frataxin has the in vitro capacity to form iron-loaded multimers, which also suggests an iron storage function. It has been reported that alanine substitution of residues in an acidic ridge of yeast frataxin (Yfh1) elicits loss of iron binding in vitro but has no effect on Fe-S cluster synthesis in vivo. Here, we show that a marked change in the electrostatic properties of a specific region of Yfh1 surface - by substituting two or four acidic residues by lysine or alanine, respectively - impairs Fe-S cluster assembly, weakens the interaction between Yfh1 and Isu1, and increases oxidative damage. Therefore, the acidic ridge is essential for the Yfh1 function and is likely to be involved in iron-mediated protein-protein interactions.

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Friedreich's ataxia is caused by a deficit in the mitochondrial protein frataxin. The present work demonstrates that in vivo yeast frataxin Yfh1p and Isu1p, the mitochondrial scaffold protein for the Fe-S cluster assembly, have tightly linked biological functions, acting in concert to promote the Fe-S cluster assembly. A synthetic lethal screen on high iron media with the mild G107D yfh1 mutant has specifically identified Isu1p. Analysis of the cellular phenotypes resulting from pairwise combinations of yfh1 and isu1 mutations, and cross-linking experiments in isolated mitochondria provide evidence for a direct interaction between Yfh1p and Isu1p.

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Yeast strain deleted for the YFH1 gene, which encodes the orthologue of human frataxin, accumulates iron in mitochondria, constitutively activates the high-affinity iron import system in the plasma membrane, and is sensitive to high iron media. We have performed a genetic screen for mutants of a yfh1 deleted strain with increased resistance to high levels of iron. One of the identified mutations caused the deletion of the hypervariable C-terminal region of Ras2p GTPase. The effect of ras2 mutation on the growth of yfh1 null strain was masked by the addition of caffeine. We found that the ras2 mutation does not alter the expression of the iron regulon nor prevent mitochondrial iron accumulation in a yfh1 mutant context. The double yfh1 ras2 mutant has increased mRNA levels of CIT2 gene and augmented catalase activity.

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Friedreich's ataxia is caused by a deficit in frataxin, a small mitochondrial protein of unknown function that has been conserved during evolution. Previous studies have pointed out a role for frataxin in mitochondrial iron-sulfur (Fe-S) metabolism. Here, we have analyzed the incorporation of Fe-S clusters into yeast ferredoxin imported into isolated energized mitochondria from cells grown in the presence of glycerol, an obligatory respiratory carbon source. Similar amounts of apo-ferredoxin precursor were imported into mitochondria and processed in wild-type and yfh1-deleted (delta YF111) strains. However, the incorporation of Fe-S clusters into apo-ferredoxin was significantly reduced in delta YFH1 mitochondria. The newly assembled ferredoxin was stable, excluding the possibility that the decreased incorporation was a result of increased oxidative damage. When delta YFH1 cells were grown in raffinose medium, the formation of holo-ferredoxin was low, as a consequence of the decrease in ferredoxin precursor import into mitochondria. However, the decrease in the conversion rate of apo- into holo-ferredoxin was in the same range as for glycerol-grown cells, indicating that the extent of the defect in Fe-S protein assembly is similar under different physiological conditions. These data show that frataxin is not essential for Fe-S protein assembly, but improves the efficiency of the process. The large variations observed in the activity of Fe-S cluster proteins under different physiological conditions result from secondary defects in the physiology of delta YFH1 cells.

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Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.

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The mitochondrial solute carriers Mrs3p and Mrs4p were originally isolated as multicopy suppressors of intron splicing defects. We show here that MRS4 is co-regulated with the iron regulon genes, and up-regulated in a strain deficient for Yfh1p, the yeast homologue of human frataxin. Using in vivo 55Fe cell radiolabeling we show that in glucose-grown cells mitochondrial iron accumulation is 5-15 times higher in deltaYFH1 than in wild-type strain. However, although in a deltaYFH1deltaMRS3deltaMRS4 strain, the intracellular 55Fe content is extremely high, the mitochondrial iron concentration is decreased to almost wild-type levels. Moreover, deltaYFH1deltaMRS3deltaMRS4 cells grown in high iron media do not lose their mitochondrial genome. Conversely, a deltaYFH1 strain overexpressing MRS4 has an increased mitochondrial iron content and no mitochondrial genome. Therefore, MRS4 is required for mitochondrial iron accumulation in deltaYFH1 cells. Expression of the iron regulon and intracellular 55Fe content are higher in a deltaMRS3deltaMRS4 strain than in the wild type. Nevertheless, the mitochondrial 55Fe content, a balance between iron uptake and exit, is decreased by a factor of two. Moreover, 55Fe incorporation into heme by ferrochelatase is increased in an MRS4-overexpressing strain. The function of MRS4 in iron import into mitochondria is discussed.

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Recently, our knowledge of yeast mitochondrial biogenesis has considerably progressed. This concerns the import machinery that guides preproteins synthesized on the cytoplasmic ribosomes through the mitochondrial outer and inner membranes, as well as the inner membrane insertion machinery of mitochondrially encoded polypeptides, or the proteins participating in the assembly and quality control of the respiratory complexes and ATP synthase. More recently, two new fields have emerged, biosynthesis of the iron-sulfur clusters and dynamics of the mitochondrion. Many of the newly discovered yeast proteins have homologues in human mitochondria. Thus, Saccharomyces cerevisiae has proven a particularly suitable simple organism for approaching the molecular bases of a growing number of human mitochondrial diseases caused by mutations in nuclear genes identified by positional cloning.

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