Yme2p is a mediator of nucleoid structure and number in mitochondria of the yeast Saccharomyces cerevisiae

A large number of gene products have been identified that either directly or indirectly alter the inheritance of mitochondrial DNA. In yeast, we have used a unique genetic screen based on the transfer of DNA from mitochondria to nucleus to identify nuclear-encoded gene products that are targeted to mitochondria and impact the stable inheritance of mitochondrial DNA. A specific allele of one of these genes, yme2-4, prevents even the low wild-type rate of mitochondrial DNA transfer to the nucleus and imparts significant temperature-sensitive and respiratory-growth defects. Intra- and extragenic suppressors of the yme2-4 growth phenotypes were isolated and analysis of these interacting genes reveals that both YME2 and its suppressors influence the structure and number of mitochondrial nucleoids. The yme2-4 allele decreases the average number of mtDNA nucleoids found in cells and the sensitivity of DNA in toluene-treated mitochondria to digestion by DNA exonuclease, effects reversed by intra- and extragenic suppressors. The extragenic suppressor, a missense allele of ILV5, encodes an enzyme of the branched-chain amino acid biosynthetic pathway that is also a component of mitochondrial nucleoids. A null allele of ILV5 suppresses transfer of mitochondrial DNA to the nucleus and displays synthetic interactions with yme2-4.     

Recombination of yeast mitochondrial DNA does not require mitochondrial protein synthesis

Summary Mitochondrial genes recombine extensively in yeast zygotes. In heteropolar crosses (⁺ × ^–) in which the ^– allele consists of an insertion, there is preferential recovery of ⁺ and markers closely linked to it. This polarity has been postulated to be a consequence of one-way gene conversion beginning at the locus (- to ⁺). We have shown that most or all mitochondrial recombination in homopolar and heteropolar crosses, and the phenomenon of polarity itself, does not require products of protein synthesis on mitochondrial ribosomes. (i) Yeast strains were grown and mated, and the zygotes plated and grown, on glucose medium with erythromycin to inhibit and dilute out the products of mitochondrial protein synthesis. Recombination frequencies and polarity at the cap1 and oli1 loci were normal compared to controls in some homopolar (⁺ × ^–) and heteropolar crosses. Apparent changes in recombination frequencies and polarity were seen in other crosses but are attributable to locus-specific petite induction by erythromycin. (ii) Homopolar (⁺ × ⁺) and heteropolar crosses between pairs of petite mutants retaining the cap1, ery1, and oli1 loci also showed nearly normal recombination at the cap1 and oli1 loci, as determined by test-crossing the petite progeny. The petite mutants and zygotes cannot do mitochondria) protein synthesis. These results support the recombinational model of polarity.     

Newly synthesised DNA of high molecular weight in the yeast Saccharomyces cerevisiae

Summary Two species of newly synthesised DNA larger than average replicons have been found in yeast. Their molecular weights are 60 million and 90 million daltons respectively. The exact nature of these molecules is not certain. They may represent entirely novel species of cellular DNA or they could be concatameric replication intermediates of some particular fraction of DNA, such as mitochondrial DNA or rDNA. Alternatively they could result from the fusion of adjacent completed replicons in a small cluster.     

Assembly of the mitochondrial membrane system. Organization of yeast mitochondrial DNA in the Oli1 region

Summary The mitochondrial DNA (mtDNA) of a cytoplasmic petite mutant (DS401) of Saccharomyces cerevisiae genetically marked for the ATPase proteolipid, serine tRNA and varl genes has been characterized by restriction endonuclease analysis and DNA sequencing. The DS401 mtDNA segment is 5.3 kb long spanning the region between 79.1 and 86.8 units of the wild type genome. Most of the DS401 mtDNA consists of A+T rich sequences. In addition, however, there are ten short sequences with a high content of G+C and two sequences that have been identified as the ATPase proteolipid and the serine tRNA genes. The two genes map at 81 and 83 units and are transcribed from the same DNA strand. Even though there are other possible coding sequences in the DNA segment, none are sufficiently long to code for a gene product of the size of the varl protein. Based on the relative organization of the G+C rich clusters and genes, a model has been proposed for the processing of mitochondria) RNA. This model postulates the existence of mitochondrial double strand specific RNases that cleave the RNA at the G+C clusters.     

Second-site antibiotic resistance mutations in the ribosomal region of yeast mitochondrial DNA

Summary A large proportion of the spontaneous erythromycin resistant mutants isolated from a strain carrying a previously-induced chloramphenicol resistance mutation at cap3 do not map at ery1, the locus most often associated with mitochondrial erythromycin resistance. Most of the new mutations are also nonallelic at spil, spi2, and other known antibiotic resistance loci within the 21S rRNA gene; they are allelic with each other and define the new locus, ery2. Induced second-site erythromycin resistant mutants from the cap ^r3 strain, as well as spontaneous or induced mutants from strains carrying a cap ^r 1 mutation, all tend to map at eryl. The cap ^r3 mutation is apparently necessary for the expression of erythromycin resistance resulting from a second mutation at ery2.     

The effect of hydroxyurea on the mechanism of DNA synthesis in the yeast Saccharomyces cerevisiae

Summary Newly synthesised DNA molecules the same size as replicons (7 million-60 million daltons) accumulate in yeast cells treated with hydroxyurea. During prolonged incubation in low concentrations of the drug, there is a large accumulation of these molecules without any corresponding increase in their molecular weight. On release from the inhibtion the molecules are converted to large molecular weight DNA. These observations are consistent with an inhibition by hydroxyurea of the joining of completed replicons. In addition, newly synthesised DNA molecules the size of yeast Okazaki fragments also accumulate in cells treated with hydroxyurea.     

Control of recombination within and between DNA plasmids of Saccharomyces cerevisiae

Summary [2 m⁺ and [2m°] yeast were transformed to stable leucine prototrophy with the hybrid yeast — E. coli plasmid, pJDB219. This plasmid contains the entire sequence of the endogenous 2 m yeast DNA plasmid in addition to the yeast nuclear LEU2 ⁺ gene and the Co1E1 derivative, pMB9. In the [2 m⁺] transformants, a new wholly yeast LEU2 ⁺ plasmid, pYX, was generated, probably by a recombination event between pJDB219 and 2 m DNA. The plamid, pYX, in the absence of 2 m DNA, was found to exist in equimolar amounts of two forms, A and B, which probably arise by intramolecular recombination across the inverted repeat sequences of the 2 m DNA portion of the plasmid. pJDB219 was found to require the presence of 2 m DNA to undergo this intramolecular recombination. The results suggest that 2, m DNA and pYX code for a gene product required in this recombination event which pJDB219 cannot produce.     

A mitochondrial frameshift suppressor maps in the tRNASer-var1 region of the mitochondrial genome of the yeast S. cerevisiae

Summary A polypeptide chain-terminating mutation (M5631) previously has been shown to be a +1T insertion in the yeast mitochondrial gene oxi1, coding for subunit II of the cytochrome c oxidase. A spontaneously arisen frameshift suppressor (mfs-1) that is mitochondrially inherited suppresses this mutation to a considerable extent. The suppressor mutation was mapped by genetic and molecular analyses in the mitochondrial tRNA^Ser-var1 region of the mitochondrial genome of the yeast S. cerevisiae. Genetic analyses show that the suppressor mfs-1 does not suppress other known mitochondrial frameshift mutations, or missense and nonsense mutations.     

The regulation of mitochondrial DNA levels in Saccharomyces cerevisiae

Summary Mitochondrial DNA (mtDNA) synthesis can continue under conditions which block cell division and nuclear DNA (nDNA) synthesis, producing cells with several times the normal level of mtDNA. We have examined mtDNA synthesis in cultures recovering from such cell cycle blocks. Our results show that the rate of mtDNA synthesis is not affected either during a block of the cell cycle with -factor or during recovery from a perturbation in the amount of mtDNA/cell induced by blocking the cell cycle with -factor or cdc4. The normal mtDNA content was restored a period of several generations when permissive conditions were restored. These results suggest that mtDNA synthesis is coupled to cell growth.     

Evolutionarily recent transfer of a group I mitochondrial intron to telomere regions in Saccharomyces cerevisiae

Summary The junctions between X and Y subtelomeric repeats in Saccharomyces cerevisiae usually contain a stretch of telomere sequences, (G_1–3T)_n. Two of three cloned X-Y junctions from strain YP1 have a replacement of about 200 bp of X, the internal telomere sequence, and 49 bp of Y by a 292 bp sequence. The first 227 bp of this insertion sequence are 100% identical to the fourth intron of cytochrome b. The rest of the insertion has homology to an unknown dispersed nuclear sequence. Recombination among subtelomeric regions can explain the nuclear distribution of this sequence and why telomeres can trap and maintain sequences that would otherwise be lost.     

Genetic control of plasmid DNA double-strand gap repair in yeast,Saccharomyces cerevisiae

Summary The repair of double-strand gaps (DSGs) in the plasmid DNA of radiosensitive mutants of Saccharomyces cerevisiae has been analyzed. The proportion of repair events that resulted in complete plasmid DNA DSG recovery was close to 100% in Rad⁺ cells. Mutation rad55 does not influence the efficiency and preciseness of DSG repair. The mutant rad57, which is capable of recombinational DNA DSB repair, resulted in no DSG recovery. Mutation rad53 substantially inhibits the efficiency of DSG repair but does not influence the precision of repair. Plasmid DNA DSG repair is completely blocked by mutations rad50 and rad54.     

Nuclear suppressors of the mitochondrial mutation oxi1-V25 in Saccharomyces cerevisiae

Summary Ten nuclear suppressors (nam mutations) of the mitochondrial oxi1-V25 ochre mutation are characterized. They restore to some extent the functional form of cytochrome oxidase, as judged by the results of growth tests, cytochrome spectra, cytochrome oxidase activities, and electrophoresis of the products of mitochondrial translation. The nam mutants can suppress some mit ^– mutations mapping in four mitochondrial genes. They act on a number of chain-terminating mit ^– mutations. When grown on glycerol medium some double mutants nam _x-V25 show an increased sensitivity to paromomycin, while the growth of others is stimulated by the drug. The nam mutants are probably omnipotent suppressors resulting from mutations in nuclear gene(s) specifying mitoribosomal protein(s).     

A mitochondrial frameshift-suppressor (+) of the yeast S. cerevisiae maps in the mitochondrial 15S rRNA locus

Summary The first case of a +1 extrageneic frameshift suppressor (MF1), mapping in the yeast mitochondrial 15S rRNA gene is reported. The suppressor was identified by genetic analyses in a leaky mitochondrial oxi1 frameshift mutant and the respective wild-type strain 777-3A of the yeast S. cerevisiae. This is in accordance with the finding that all mitochondrial frameshift mutants isolated from this strain tend to be leaky to a variable degree. MF1 does not suppress known nonsense mutations created by a direct basepair exchange in strain 777-3A. These mutants exhibit a non-leaky phenotype (Weiss-Brummer et al. 1984).     

YNT20, a bypass suppressor of yme1 yme2, encodes a putative 3′-5′ exonuclease localized in mitochondria of Saccharomyces cerevisiae

Mutation of YME genes in yeast results in a high rate of mitochondrial DNA escape to the nucleus. The synthetic respiratory growth defect of yme1 yme2 yeast strains is suppressed by recessive mutations in YNT20. Inactivation of YNT20 creates a cold-sensitive respiratory growth defect that is more pronounced in a yme1 background and which is suppressed by yme2. Inactivation of YNT20 causes a qualitative reduction in the rate of mitochondrial DNA escape in yme1, but not yme2, strains, suggesting that YNT20 plays a role in the yme1-mediated mitochondrial DNA escape pathway. YNT20p is a soluble mitochondrial protein that belongs to a subfamily of putative 3′-5′ exonucleases. Furthermore, conserved sequence elements in Yme2p suggest that this protein may also function as an exonuclease. Received: 26 August / 13 October 1998     

Helicase Hmi1 stimulates the synthesis of concatemeric mitochondrial DNA molecules in yeast Saccharomyces cerevisiae

Hmi1p is a helicase in the yeast Saccharomyces cerevisiae required for maintenance of the wild-type mitochondrial genome. Disruption of the HMI1 ORF generates ^– and ⁰ cells. Here we demonstrate that, in ^– yeast strains, Hmi1p stimulates the synthesis of long concatemeric mitochondrial DNA molecules associated with a reduction in the number of nucleoids used for mitochondrial DNA packaging. Surprisingly, the ATPase negative mutants of Hmi1p can also stimulate the synthesis of long concatemeric ^– mitochondrial DNA molecules and support the maintenance of the wild-type mitochondrial genome, albeit with reduced efficiency. We show that, in the mutant hmi1–5 background, the wild-type mitochondrial DNA is fragmented; and we propose that, in hmi1 yeast cells, the loss of the wild-type mitochondrial genome is caused by this fragmentation of the mitochondrial DNA.     

Preparation of yeast mitochondrial DNA for direct sequence analysis

We describe two simple protocols for preparation of templates for direct sequencing of yeast mitochondrial DNA (mtDNA) by automatic DNA analyzers. The protocols work with a range of yeast species and yield a sufficient quantity and quality of the template DNA. In combination with primer-walking strategy, they can be used either as an alternative or a complementary approach to shot-gun sequencing of random fragment DNA libraries. We demonstrate that the templates are suitable for re-sequencing of the mtDNA for comparative analyses of intraspecific variability of yeast strains as well as for primary determination of the complete mitochondrial genome sequence.