Assembly of the Mitochondrial Membrane System: Nuclear Suppression of a Cytochrome b Mutation in Yeast Mitochondrial DNA

In a previous study, a mitochondrial mutant expressing a specific enzymatic deficiency in co-enzyme QH₂-cytochrome c reductase was described (Tzagoloff, Foury and Akai 1976). Analysis of the mitochondrially translated proteins revealed the absence in the mutant of the mitochondrial product corresponding to cytochrome b and the presence of a new low molecular weight product. The premature chain-termination mutant was used to obtain suppressor mutants with wild-type properties. One such revertant strain was analyzed genetically and biochemically. The revertant was determined to have a second mutation in a nuclear gene that is capable of partially suppressing the original mitochondrial cytochrome b mutation. Genetic data indicate that the nuclear mutation is recessive and is probably in a gene coding for a protein involved in the mitochondrial translation machinery.     

Mechanism of Mitochondrial Mutation in Yeast

THE yeast Saccharomyces cerevisiae can mutate to the respiratory-incompetent petite colony form. The mutation is probably caused by damage to, or loss of, the yeast's mitochondrial DNA, for petite mutants often lack mitochondrial DNA, possess it in abnormal amounts or with abnormal buoyant density¹. Some of the agents, such as acrifiavine or ethidium bromide, which induce the petite mutation interfere with mitochondrial DNA synthesis^2,3 whereas ethidium bromide also causes or permits degradation of Saccharomyces cerevisiae mitochondrial DNA^2,3. We have observed that nalidixate (50 µg/ml.), an inhibitor of DNA synthesis, can prevent or delay petite mutation induced by ethidium bromide⁴. A similar effect has been observed by Hollenberg and Borst using a higher nalidixate concentration⁵. We have investigated the mechanism of this effect. A diploid prototrophic strain of Saccharomyces cerevisiae (NCYC 239) was used throughout.     

A Cytoplasmic Suppressor of a Nuclear Mutation Affecting Mitochondrial Functions in Drosophila

Phenotypes relevant to oxidative phosphorylation (OXPHOS) in eukaryotes are jointly determined by nuclear and mitochondrial DNA (mtDNA). Thus, in humans, the variable clinical presentations of mitochondrial disease patients bearing the same primary mutation, whether in nuclear or mitochondrial DNA, have been attributed to putative genetic determinants carried in the “other” genome, though their identity and the molecular mechanism(s) by which they might act remain elusive. Here we demonstrate cytoplasmic suppression of the mitochondrial disease-like phenotype of the Drosophila melanogaster nuclear mutant tko^25t, which includes developmental delay, seizure sensitivity, and defective male courtship. The tko^25t strain carries a mutation in a mitoribosomal protein gene, causing OXPHOS deficiency due to defective intramitochondrial protein synthesis. Phenotypic suppression was associated with increased mtDNA copy number and increased mitochondrial biogenesis, as measured by the expression levels of porin voltage dependent anion channel and Spargel (PGC1α). Ubiquitous overexpression of Spargel in tko^25t flies phenocopied the suppressor, identifying it as a key mechanistic target thereof. Suppressor-strain mtDNAs differed from related nonsuppressor strain mtDNAs by several coding-region polymorphisms and by length and sequence variation in the noncoding region (NCR), in which the origin of mtDNA replication is located. Cytoplasm from four of five originally Wolbachia-infected strains showed the same suppressor effect, whereas that from neither of two uninfected strains did so, suggesting that the stress of chronic Wolbachia infection may provide evolutionary selection for improved mitochondrial fitness under metabolic stress. Our findings provide a paradigm for understanding the role of mtDNA genotype in human disease.     

A Mutation in a Novel Yeast Proteasomal Gene, RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.     

Effects of a Mitochondrial Mutator Mutation in Yeast POS5 NADH Kinase on Mitochondrial Nucleotides

Saccharomyces cerevisiae contains three NADH/NAD(+) kinases, one of which is localized in mitochondria and phosphorylates NADH in preference to NAD(+). Strand et al. reported that a yeast mutation in POS5, which encodes the mitochondrial NADH kinase, is a mutator, specific for mitochondrial genes (Strand, M. K., Stuart, G. R., Longley, M. J., Graziewicz, M. A., Dominick, O. C., and Copeland, W. C. (2003) Eukaryot. Cell 2, 809-820). Because of the involvement of NADPH in deoxyribonucleotide biosynthesis, we asked whether mitochondria in a pos5 deletion mutant contain abnormal deoxyribonucleoside triphosphate (dNTP) pools. We found the pools of the four dNTPs to be more than doubled in mutant mitochondrial extracts relative to wild-type mitochondrial extracts. This might partly explain the mitochondrial mutator phenotype. However, the loss of antioxidant protection is also likely to be significant. To this end, we measured pyridine nucleotide pools in mutant and wild-type mitochondrial extracts and found NADPH levels to be diminished by ～4-fold in Δpos5 mitochondrial extracts, with NADP(+) diminished to a lesser degree. Our data suggest that both dNTP abnormalities and lack of antioxidant protection contribute to elevated mitochondrial gene mutagenesis in cells lacking the mitochondrial NADH kinase. The data also confirm previous reports of the specific function of Pos5p in mitochondrial NADP(+) and NADPH biosynthesis.     

A Genome-Wide Map of Mitochondrial DNA Recombination in Yeast

In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome maintenance.     

线粒体基因突变与NIDDM发生的关系

采用PCR-SSCP、PCR-RFLP及PCR产物直接测序等技术对90例NIDDM(即非胰岛素依赖型糖尿病)及80例正常对照个体的血细胞线粒体DNA进行了突变分析。结果在2例患者中发现线粒体DNA(mitochondrial DNA,mtDNA) ND1 (NaDH Dehydrogenase subunitⅠ)基因上3316位点存在G→A的点突变,导致丙氨酸错义突变成苏氨酸,而在80例正常对照个体中均不存在此位点突变。国内外已证实的和1.5%NIDDM发生有关的mtDNA tRNA Leu^(UUR)|基因上3243位点A→G的突变在本实验中并未发现。由此推断,3316位点G→A的突变可能与NIDDM的发生在关,3243位点A→G的突变率确实很低,可见糖尿病的发生在线粒体遗传上具有广泛的异质性。 Abstract:Using PCR-SSCP,PCR-RFLP and PCR product direct sequencing techniques,we analysed the mitochondrial DNAs(mtDNAs)of 90 patients with NIDDM (Non Insulin-Dependent Diabetes Mellitus)and those of 80 normal controls.The results showed that a G to A mutation which leads alanine’s missence mutaton to threonine in the mitochondrial ND1(NaDH Dehydrogenase subunit I) gene at nucleotide pair 3316 occurred in the blood cells of 2 patients.We have not however,indentified with the A to G mutation at nucleotide pair 3243 of the mitochondrial tRNA Leu(UUR) gene,which has been reported to associate with NIDDM in about 1.5% of the diabetic population.We infer that the mutation at position 3316 is perhaps associated with the development of NIDDM,the occurance of the mutation at position 3243 is actually rare,and NIDDM has an intensive mitochondrial genetic heterogenous background.     

Nuclear Mutations in the Petite-Negative Yeast Schizosaccharomyces Pombe Allow Growth of Cells Lacking Mitochondrial DNA

The fission yeast Schizosaccharomyces pombe has never been found to give rise to viable cells totally lacking mitochondrial DNA (rho(o)). This paper describes the isolation of rho(o) strains of S. pombe by very long term incubation of cells in liquid medium containing glucose, potassium acetate and ethidium bromide. Once isolated, the rho(o) strains did not require potassium acetate or any other novel growth factors. These nonrespiring strains contained no mitochondrial DNA (mtDNA) detectable either by gel-blot hybridization using as probe a clone containing the entire S. pombe mtDNA, or by 1',6-diamidino-2-phenylindole staining of whole cells. Induction of rho(o) derivatives of standard laboratory strains was not reproducible from culture to culture. The cause of this irreproducibility appears to be that growth of the rho(o) strains of S. pombe depended on nuclear mutations that occurred in some, but not all, of the initial cultures. Two independent rho(o) isolates contained mutations in unlinked genes, termed ptp1-1 and ptp2-1. These mutations allowed reproducible ethidium bromide induction of viable rho(o) strains. No other phenotypes were associated with ptp mutations in rho+ strains.     

线粒体DNA突变与肿瘤发生

线粒体是哺乳动物细胞内唯一含有核外遗传物质的细胞器。由于其自身的特征以及所处的环境等因素,较之于nDNA,mtDNA更容易受到损伤因子的攻击。mtDNA突变,mtDNA片段整合入核基因组等可能与肿瘤的发生以及肿瘤表型的产生有较为密切的关系。深入研究线粒体及其基因组的结构和表达调控,探讨核质关系,对于阐明细胞癌变的机制可能具有重要意义。     

Analysis of Mutation Mechanisms in Human Mitochondrial DNA

The cause of the high variability of human mitochondrial DNA (mtDNA) remains largely unknown. Three mechanisms of mutagenesis that might account for the generation of nucleotide substitutions in mtDNA have been analyzed: deamination of DNA nitrous bases caused by deamination agents, tautomeric proton migration in nitrous bases, and the hydrolysis of the glycoside bond between the nitrous base and carbohydrate residue in nucleotides against the background of the free-radical damage of DNA polymerase γ. Quantum chemical calculations demonstrated that the hydrolysis of the N-glycoside bond is the most probable mechanism; it is especially prominent in the H strand, which remains free during mtDNA replication for a relatively long time. It has also been found that hydrolytic deamination of adenine in single-stranded regions of the H strand is a possible cause of the high frequency of T → C transitions in the mutation spectra of the L-chain of the major mtDNA noncoding region.     

Mitochondrial DNA Transmission Genetics in Crickets

This paper presents the results of a single generation study of the transmission genetics of mitochondrial DNA in the field cricket Gryllus firmus. In this species, individuals heteroplasmic for at least two different-sized mitochondrial genomes can be collected easily from natural populations. The frequencies of mtDNA size variants in heteroplasmic females and samples of their offspring were estimated by densitometry of autoradiographs. The variance in mitochondrial genotype frequencies among the offspring of heteroplasmic females indicates that, through genetic drift, fixation would take several hundred animal generations. Differences between the observations and data on mtDNA transmission in yeast and cows are discussed in light of the differences in organelle sampling regime and early developmental events in these species. Our data also show shifts in genotype frequencies in the transmission from mother to offspring that suggest a bias in favor of smaller genomes. The nature of mtDNA size variation in natural populations of crickets is discussed in reference to a mutation-selection balance.     

线粒体DNA突变与氨基糖甙类抗生素致聋

近年来的研究表明,氨基糖甙类抗生素致聋与线粒体DNA突变有关。本文从分子水平阐述了线粒体DNA突变的发生情况及其在氨基糖甙类抗生素致聋中的作用。     

线粒体DNA突变相关疾病的基因治疗

mtDNA是动物细胞染色体外唯一存在的遗传物质,mtDNA突变相关疾病正越来越为人们所认识,但目前尚无有效的治疗手段。应用亲脂质阳离子,构建靶向线粒体PNA,转线粒体移植手段。以及构建mtDNA样质粒,都可能是今后的研究方向。     

Incomplete Maternal Transmission of Mitochondrial DNA in Drosophila

The possibility of incomplete maternal transmission of mitochondrial DNA (mtDNA) in Drosophila, previously suggested by the presence of heteroplasmy, was examined by intra- and interspecific backcrosses of Drosophila simulans and its closest relative, Drosophila mauritiana. mtDNAs of offspring in these crosses were characterized by Southern hybridization with two alpha-32P-labeled probes that are specific to paternal mtDNAs. This method could detect as little as 0.03% paternal mtDNA, if present, in a sample. Among 331 lines that had been backcrossed for ten generations, four lines from the interspecific cross D. simulans (female) x D. mauritiana (male) showed clear evidence for paternal leakage of mtDNA. In three of these the maternal type was completely replaced while the fourth was heteroplasmic. Since in this experiment the total number of fertilization is known to be 331 x 10 = 3310, the proportion of paternal mtDNA per fertilization was estimated as about 0.1%. The mechanisms and evolutionary significance for paternal leakage are discussed in light of this finding.     

Human Dna2 Is a Nuclear and Mitochondrial DNA Maintenance Protein

Dna2 is a highly conserved helicase/nuclease that in yeast participates in Okazaki fragment processing, DNA repair, and telomere maintenance. Here, we investigated the biological function of human Dna2 (hDna2). Immunofluorescence and biochemical fractionation studies demonstrated that hDna2 was present in both the nucleus and the mitochondria. Analysis of mitochondrial hDna2 revealed that it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids. RNA interference-mediated depletion of hDna2 led to a modest decrease in mitochondrial DNA replication intermediates and inefficient repair of damaged mitochondrial DNA. Importantly, hDna2 depletion also resulted in the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that nuclear hDna2 plays a role in genomic DNA stability. Together, our data indicate that hDna2 is similar to its yeast counterpart and is a new addition to the growing list of proteins that participate in both nuclear and mitochondrial DNA maintenance.DNA damage arises from errors in the replication process, as well as a myriad of intrinsic and extrinsic DNA-damaging agents that continually assault cells. Failure to efficiently repair DNA lesions leads to accumulation of mutations that contribute to numerous pathologies, including carcinogenesis. In addition to genomic DNA, mitochondrial DNA (mtDNA) is subject to damage that requires repair to maintain integrity. For these reasons, it is not surprising that DNA replication and repair proteins display significant plasticity that allows participation in several divergent replication and repair processes. In addition, numerous mechanisms, including alternative splicing, posttranslational modifications, or utilization of alternative translation initiation start sites, allow DNA replication and repair proteins such as Pif1, DNA ligase III, and APE1 to localize to the nucleus and the mitochondrion and participate in DNA replication and/or repair (9, 17, 25), thus ensuring genomic DNA and mtDNA integrity.Dna2 is an evolutionarily conserved helicase/nuclease enzyme. Originally discovered in Saccharomyces cerevisiae, Dna2 orthologs are found throughout the animal kingdom, including humans (5, 22, 28). Early studies demonstrated that Dna2 functions in concert with Flap endonuclease 1 (FEN1) to remove long DNA flaps that form upon lagging-strand DNA replication (6). However, in contrast to FEN1, Dna2 is an essential gene in yeast, suggesting that other proteins, including FEN1, cannot compensate for its loss in DNA replication or that it possesses functions beyond its role in Okazaki fragment processing. In agreement with this, genetic and biochemical studies have implicated Dna2 in DNA double-strand break (DSB) repair, telomere regulation, and mitochondrial function (8, 10, 15, 26, 38, 44, 45).Analysis of Dna2 in yeast revealed that it undergoes dynamic cell cycle localization. Dna2 localizes to telomeres during G₁, relocalizes throughout the genome in S phase, and moves back to the telomere during late S/G₂, where it participates in telomere replication and telomerase-dependent telomere elongation (10). Dna2 also leaves the telomere following treatment with bleomycin and localizes to sites of DNA DSBs (10). In addition, dna2 mutants are sensitive to DNA damage induced by gamma radiation and methanesulfonic acid methyl ester (7, 15). These phenotypes may be explained by recent work demonstrating that Dna2 plays an important role in 5′-end resection following DSBs. Indeed, upon induction of DSBs and initiation of 5′-end resection by the Mre11-Rad50-Xrs2 complex, Dna2 and Sgs1 cooperate to further degrade the 5′ end, creating long 3′ strands essential for homologous recombination (26, 45). Finally, while dna2Δ mutations are lethal in budding yeast, the dna2Δ pif1-m2 (nuclear PIF1) double mutations rescue dna2Δ lethality but produce a petite phenotype, suggesting that Dna2 is also involved in mtDNA maintenance (8).Recently, the human ortholog of Dna2 was cloned and characterized (23, 29). Biochemical analysis revealed that, similar to its yeast counterpart, the human Dna2 (hDna2) protein possesses nuclease, ATPase, and limited helicase activities (23, 29), suggesting that it carries out analogous functions in yeast and mammalian cells. However, hDna2''s putative role in genomic DNA repair and replication was called into question by a recent study suggesting that hDna2 is absent from the nucleus and found exclusively within the mitochondria, where it participates in mtDNA repair (44). Further in vitro biochemical studies suggested that hDna2 also participates in mtDNA replication (44). Here, we confirm that hDna2 localizes to the mitochondria and demonstrate that hDna2 participates in mtDNA replication and repair. However, our studies go further by uncovering a nuclear form of hDna2 that plays an important role in genomic stability. Indeed, we demonstrate that depletion of hDna2 leads to the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that hDna2, like its yeast counterpart, is essential to maintain nuclear DNA stability.     

On the Mutation Rates of the Mitochondrial and Nuclear Genomes of Salmonid Fishes

Mutation rates of the mitochondrial and nuclear genomes of salmonid fishes were assessed on the basis of a phylogenetic study of 12 species representing four genera of the family Salmonidae. Analysis of the extent of divergence of the masu salmon Oncorhynchus masou and the Pacific trout Parasalmo suggests a high rate of mtDNA mutation in the masu salmon. However, the nuclear genome in this species has mutated relatively slowly. For the other 5 species of Pacific salmon, no discrepancy was found in the mutation rates of mitochondrial and nuclear DNA. Values of the absolute time of divergence of taxa, calculated for the two independently inherited parts of the salmonid genome, were approximately within the same range and coincided with those based on evolutionary hypotheses [1, 21].     

A New Method of the Visualization of the Double-Stranded Mitochondrial and Nuclear DNA

The study describes the method of a sensitive detection of double-stranded DNA molecules in situ. It is based on the oxidative attack on the deoxyribose moiety by copper(I) in the presence of oxygen. We have shown previously that the oxidative attack leads to the formation of frequent gaps in DNA. Here we have demonstrated that the gaps can be utilized as the origins for an efficient synthesis of complementary labeled strands by DNA polymerase I and that such enzymatic detection of the double-stranded DNA is a sensitive approach enabling in-situ detection of both the nuclear and mitochondrial genomes in formaldehyde-fixed human cells.     

Mutations in the Mitochondrial Atp Synthase Gamma Subunit Suppress a Slow-Growth Phenotype of Yme1 Yeast Lacking Mitochondrial DNA

In Saccharomyces cerevisiae, inactivation of the nuclear gene YME1 causes several phenotypes associated with impairment of mitochondrial function. In addition to deficiencies in mitochondrial compartment integrity and respiratory growth, yme1 mutants grow extremely slowly in the absence of mitochondrial DNA. We have identified two genetic loci that, when mutated, act as dominant suppressors of the slow-growth phenotype of yme1 strains lacking mitochondrial DNA. These mutations only suppressed the slow-growth phenotype of yme1 strains lacking mitochondrial DNA and had no effect on other phenotypes associated with yme1 mutations. One allele of one linkage group had a collateral respiratory deficient phenotype that allowed the isolation of the wild-type gene. This suppressing mutation was in ATP3, a gene that encodes the gamma subunit of the mitochondrial ATP synthase. Recovery of two of the suppressing ATP3 alleles and subsequent sequence analysis placed the suppressing mutations at strictly conserved residues near the C terminus of Atp3p. Deletion of the ATP3 genomic locus resulted in an inability to utilize nonfermentable carbon sources. atp3 deletion strains lacking mitochondrial DNA grew slowly on glucose media but were not as compromised for growth as yme1 yeast lacking mitochondrial DNA.     

Maintenance and Integrity of the Mitochondrial Genome: a Plethora of Nuclear Genes in the Budding Yeast

Instability of the mitochondrial genome (mtDNA) is a general problem from yeasts to humans. However, its genetic control is not well documented except in the yeast Saccharomyces cerevisiae. From the discovery, 50 years ago, of the petite mutants by Ephrussi and his coworkers, it has been shown that more than 100 nuclear genes directly or indirectly influence the fate of the rho⁺ mtDNA. It is not surprising that mutations in genes involved in mtDNA metabolism (replication, repair, and recombination) can cause a complete loss of mtDNA (rho⁰ petites) and/or lead to truncated forms (rho⁻) of this genome. However, most loss-of-function mutations which increase yeast mtDNA instability act indirectly: they lie in genes controlling functions as diverse as mitochondrial translation, ATP synthase, iron homeostasis, fatty acid metabolism, mitochondrial morphology, and so on. In a few cases it has been shown that gene overexpression increases the levels of petite mutants. Mutations in other genes are lethal in the absence of a functional mtDNA and thus convert this petite-positive yeast into a petite-negative form: petite cells cannot be recovered in these genetic contexts. Most of the data are explained if one assumes that the maintenance of the rho⁺ genome depends on a centromere-like structure dispensable for the maintenance of rho⁻ mtDNA and/or the function of mitochondrially encoded ATP synthase subunits, especially ATP6. In fact, the real challenge for the next 50 years will be to assemble the pieces of this puzzle by using yeast and to use complementary models, especially in strict aerobes.