On the mechanism of premeiotic DNA synthesis in the yeast Saccharomyces cerevisiae

Premeiotic DNA synthesis in synchronously sporulating cultures of the yeast, Saccharomyces cerevisiae, was analysed by sedimentation in alkaline sucrose gradients and by DNA-fibre autoradiography. The gradient profiles of cells pulse-labelled for varying times were essentially identical with those obtained with mitotic cultures, revealing a close resemblance between the meiotic and mitotic replication mechanisms. This was supported by the finding that exposure of meiotic cells to a specific concentration of hydroxyurea led to the accumulation of completed, but unjoined replicons, just as it does in mitotic cells. The results of DNA-fibre autoradiography confirmed that replicons in meiotic cells are the same size (20–180 Kb, averaging around 90 Kb) as in mitotic cells, and assuming replication is bi-directional, replication forks must move at round the same rate as in mitosis, i.e. about 0.7 μm/min.     

Excision repair of DNA in nuclear extracts from the yeast Saccharomyces cerevisiae.

Excision repair of DNA is an important cellular response to DNA damage caused by a broad spectrum of physical and chemical agents. We have established a cell-free system in which damage-specific DNA repair synthesis can be demonstrated in vitro with nuclear extracts from the yeast Saccharomyces cerevisiae. Repair synthesis of UV-irradiated plasmid DNA was observed in a radiation dose-dependent manner and was unaffected by mutations in the RAD1, RAD2, RAD3, RAD4, RAD10, or APN1 genes. DNA damaged with cis-platin was not recognized as a substrate for repair synthesis. Further examination of the repair synthesis observed with UV-irradiated DNA revealed that it is dependent on the presence of endonuclease III-sensitive lesions in DNA, but not pyrimidine dimers. These observations suggest that the repair synthesis observed in yeast nuclear extracts reflects base excision repair of DNA. Our data indicate that the patch size of this repair synthesis is at least seven nucleotides. This system is expected to facilitate the identification of specific gene products which participate in base excision repair in yeast.     

Formamidopyrimidine DNA glycosylase in the yeast Saccharomyces cerevisiae.

A DNA glycosylase that excises, 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) from double stranded DNA has been purified 28,570-fold from the yeast Saccharomyces cerevisiae. Gel filtration chromatography shows that yeast Fapy DNA glycosylase has a molecular weight of about 40 kDa. The Fapy DNA glycosylase is active in the presence of EDTA, but is completely inhibited by 0.2 M KCl. Yeast Fapy DNA glycosylase does not excise N7-methylguanine, N3-methyladenine or uracil. A repair enzyme for 7,8-dihydro-8-oxoguanine (8-OxoG) co-purifies with the Fapy DNA glycosylase. This repair activity causes strand cleavage at the site of 8-OxoG in DNA duplexes. The highest rate of incision of the 8-OxoG-containing strand was observed for duplexes where 8-OxoG was opposite guanine. The mode of incision at 8-OxoG was not established yet. The results however suggest that the Fapy- and 8-OxoG-repair activities are associated with a single protein.     

The isolation of new DNA synthesis mutants in the yeast Saccharomyces cerevisiae

Summary Sixty-eight new conditional cell cycle mutants have been isolated on the basis of their terminal cellular morphology (dumbbells). Fifteen mutants falling into nine complementation groups, were grossly defective in DNA replication and have been assigned the provisional gene symboldbf (fordumbbellformer). Dbf1 and2 stop DNA synthesis immediately on transfer to 37°C and are presumably defective in enzymes required for polymerization. Neither, however, possess a thermolabile DNA polymerase A or B.Dbf3 and4 show a pattern of synthesis consistent with their being deficient in initiation of DNA synthesis. This is confirmed in the accompanying paper.The remaining mutants are deficient in the synthesis of RNA as well as DNA. Indeed the four members of one complementation group are allelic withrna3, one of the group of mutants originally isolated as defective in RNA synthesis, and which do not exhibit a cell cycle phenotype. A re-examination of this group of mutants however, showed the bulk of them also to be defective in DNA synthesis. Furthermore, in preliminary experimentsrna3 and our four new alleles of it, together withrna6 anddbf5 and6, showed enhanced spontaneous mutation frequency.     

Mitochondrial DNA synthesis in synchronous cultures of the yeast,Saccharomyces cerevisiae

The synthesis of mitochondrial DNA (mtDNA) has been investigated by three independent methods of analysis during consecutive synchronous cell cycles in the yeast, Saccharomyces cerevisiae. The rates of pulse-label incorporation indicate maximal [³H]adenine uptake into mtDNA at the time of nuclear DNA synthesis. In contrast, the relative concentrations of mtDNA as determined by both the ratio of mtDNA to total cellular DNA and by the kinetics of isotope dilution analysis were found to increase continuously during synchronous growth. We conclude that whereas nuclear DNA replicates discontinuously during the cell cycle, mitochondrial DNA is synthesized continuously during this time. The discontinuous pattern of pulse-label incorporation into mtDNA is not considered to reflect its true mode of replication during the cell cycle.     

Determination of the nuclear DNA content of Saccharomyces cerevisiae and implications for the organization of DNA in yeast chromosomes.

The DNA content of the nucleus of the yeast Saccharomyces cerevisiae has been determined by both renaturation kinetics and DNA per cell measurements. Renaturation kinetics experiments were performed by following the decrease of optical hyperchromicity at 260 nm and by hydroxyapatite chromatography. DNA per cell measurements were made by the diaminobenzoic acid method and by the ethidium bromide method of Klotz &; Zimm (1972b). The conclusion from the above experiments is that the S. cerevisiae nucleus contains 9 × 10⁹ ± 2 × 10⁹ daltons of DNA. Previously we (Lauer &; Klotz, 1975) had measured the molecular weight of the largest piece of DNA in the yeast nucleus to be 2 × 10⁹ ± 0.2 × 10⁹. Here we extend this work by using a more highly protein-denaturing buffer system and conclude that the largest piece of DNA in the S. cerevisiae nucleus contains 1.5 × 10⁹ to 2.2 × 10⁹ daltons of DNA in both haploid and diploid cell lysates. From genetics, the largest yeast chromosome should contain 13% of the genome, or 0.9 × 10⁹ to 1.5 × 10⁹ daltons of DNA (using our DNA per cell range). Thus, the large DNA we measure contains from one to two times the amount of the DNA predicted from genetics to be in the largest chromosome. In light of these new data, viscoelastic measurements on yeast DNA are now consistent with the idea that each chromosome contains one piece of DNA.     

A DNA integrity network in the yeast Saccharomyces cerevisiae

A network governing DNA integrity was identified in yeast by a global genetic analysis of synthetic fitness or lethality defect (SFL) interactions. Within this network, 16 functional modules or minipathways were defined based on patterns of global SFL interactions. Modules or genes involved in DNA replication, DNA-replication checkpoint (DRC) signaling, and oxidative stress response were identified as the major guardians against lethal spontaneous DNA damage, efficient repair of which requires the functions of the DNA-damage checkpoint signaling and multiple DNA-repair pathways. This genome-wide genetic interaction network also identified novel components (DIA2, NPT1, HST3, HST4, and the CSM1 module) that potentially contribute to mitotic DNA replication and genomic stability and revealed novel functions of well-studied genes (the CTF18 module) in DRC signaling. This network will guide more detailed characterization of mechanisms governing DNA integrity in yeast and other organisms.     

Cytochrome synthesis in synchronous cultures of the yeast, Saccharomyces cerevisiae.

The synthesis of cytochromes aa3, b, and c has been investigated during synchronous growth in the yeast, Saccharomyces cerevisiae. These cytochromes increase in concentration continuously throughout each cell cycle, with an approximate doubling in rate during successive cycles. The rates of cytochrome formation are considerably higher in galactose-grown cultures than in cells grown in glucose. Although cytochrome aa3 increases at a continuous rate, its functional counterpart, cytochrome c oxidase, increases in stepwise fashion, with the increments occurring at the beginning of each new cell cycle. Chloramphenicol, a specific inhibitor of intramitochondrial protein synthesis, inhibits the formation of cytochrome aa3 at all stages of the cell cycle, but does not inhibit cytochrome c. Chloramphenicol exhibits a somewhat intermediate effect on cytochrome b synthesis, with transient inhibition occurring only when the drug is added prior to or during the initial part of the first cell cycle. After this time, chloramphenicol had no effect on the rate of cytochrome b synthesis. The data indicate that under our conditions of cell synchrony mitochondrial membrane formation as reflected by increments in mitochondrial cytochromes occurs by continuous accretion of new material throughout the cell cycle. Intramitochondrially synthesized polypeptide products, responsible for the formation of new cytochrome aa3, appear to be synthesized throughout the cell cycle.     

A particulate form of alkaline phosphatase in the yeast,Saccharomyces cerevisiae

A new form of alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) has been identified in the yeast Saccharomyces cerevisiae. Utilizing either synthetic or natural substrates, the enzyme exhibited a broad pH activity curve with maximum activity between 8.5 and 9.0. The enzyme was nonspecific with respect to substrate, attacking a variety of compounds containing phosphomonoester linkages, but has no detectable activity against polyphosphate, pyrophosphate or phosphodiester linkages. The enzyme exhibited an apparent K_m of 0.25 mM with respect to p-nitrophenyl phosphate, 0.38 mM with respect to α-naphthyl phosphate, and 1.0 mM with respect to 5′ AMP. The enzyme is regulated in a constitutive manner and its activity does not increase during phosphate starvation or sporulation, as does the repressible alkaline phosphatase. The enzyme is tightly bound to a particulate fraction of the cell, tentatively identified as the tonoplast membrane. It is not solubilized by treatment with high concentrations of NaCl, KH₂PO₄ or chaotropic agents. Triton X-100 (0.1%) solubilizes 12% of the particulate activity. This enzyme is differentiated from the other alkaline phosphatases found in yeast by its chromatographic elution from DEAE-cellulose, kinetic parameters, heat stability and pH stability, as well as its particulate nature. This particulate alkaline phosphatase was found in every strain examined. It has a significantly lower specific activity in the phoH mutant and a higher activity in the acid phosphatase constitutive mutant A137.     

An in vivo-in vitro alkaline DNA unwinding assay for hepatic DNA damage: comparison with the alkaline sucrose gradient centrifugation technique

The addition of glycerol, sucrose, or other diol-containing reagents to solutions of aminoacyl-tRNA (aa-tRNA) substantially increased the rate of hydrolysis of the aminoacyl ester bond. Glycerol at 4.9% (v/v) doubled the rate of deacylation for several aa-tRNAs and peptidyl-tRNAs, including fMet-tRNAMetf, while 1% (v/v) glycerol increased the deacylation rate by 20%. This effect was not caused by a nuclease contamination, and tRNA deacylated in the presence of glycerol could be fully recharged. The deacylation of aa-tRNA was accelerated by glycerol and sucrose even in the presence of EF-Tu X GTP. In addition, the extent of tRNA aminoacylation was reduced when glycerol was present at concentrations above 2% (v/v). Thus, glycerol and sucrose are not necessarily inert or neutral additions to an in vitro incubation.     

Eukaryotic DNA repair: glimpses through the yeast Saccharomyces cerevisiae.

Eukaryotic cells are able to mount several genetically complex cellular responses to DNA damage. The yeast Saccharomyces cerevisiae is a genetically well characterized organism that is also amenable to molecular and biochemical studies. Hence, this organism has provided a useful and informative model for dissecting the biochemistry and molecular biology of DNA repair in eukaryotes.     

Nucleolar and nuclear envelope proteins of the yeast Saccharomyces cerevisiae

We have developed a fast and reliable purification protocol to obtain yeast nuclei in intact and pure form and in a reasonable yield. The purified nuclei appear homogeneous at the light and electron microscopic level, are highly enriched in the nuclear marker histone H2B and devoid of mitochondrial, vacuolar and cytosolic marker proteins. On sodium dodecyl sulfate (SDS)-polyacrylamide gels, the nuclear fraction contains unique proteins which distinguishes them from the major yeast subcellular fractions. Yeast nuclei were separated by detergent/salt extraction into soluble, insoluble and membrane fractions. Antibodies raised against subnuclear fractions lead to the identification of an integral nuclear membrane protein and a high-abundance 38-kDa protein which is located in the yeast nucleolus.     

Molecular mechanism of autophagy in yeast, Saccharomyces cerevisiae

Bulk degradation of cytosol and organelles is important for cellular homeostasis under nutrient limitation, cell differentiation and development. This process occurs in a lytic compartment, and autophagy is the major route to the lysosome and/or vacuole. We found that yeast, Saccharomyces cerevisiae, induces autophagy under various starvation conditions. The whole process is essentially the same as macroautophagy in higher eukaryotic cells. However, little is known about the mechanism of autophagy at a molecular level. To elucidate the molecules involved, a genetic approach was carried out and a total of 16 autophagy-defective mutants (apg) were isolated. So far, 14 APG genes have been cloned. Among them we recently found a unique protein conjugation system essential for autophagy. The C-terminal glycine residue of a novel modifier protein Apg12p, a 186-amino-acid protein, is conjugated to a lysine residue of Apg5p, a 294-amino-acid protein, via an isopeptide bond. We also found that apg7 and apg10 mutants were unable to form an Apg12p-Apg5p conjugate. The conjugation reaction is mediated via Apg7p, E1-like activating enzyme and Apg10p, indicating that it is a ubiquitination-like system. These APG genes have mammalian homologues, suggesting that the Apg12 system is conserved from yeast to human. Further molecular and cell biological analyses of APG gene products will give us crucial clues to uncover the mechanism and regulation of autophagy.     

Modulation of DNA synthesis in Saccharomyces cerevisiae nuclear extract by DNA polymerases and the origin recognition complex

We have analyzed the modulation of DNA synthesis on a supercoiled plasmid DNA template by DNA polymerases (pol), minichromosome maintenance protein complex (Mcm), topoisomerases, and the origin recognition complex (ORC) using an in vitro assay system. Antisera specific against the four-subunit pol alpha, the catalytic subunit of pol delta, and the Mcm467 complex each inhibited DNA synthesis. However, DNA synthesis in this system appeared to be independent of polepsilon. Consequently, DNA synthesis in the in vitro system appeared to depend only on two polymerases, alpha and delta, as well as the Mcm467 DNA helicase. This system requires supercoiled plasmid DNA template and DNA synthesis absolutely required DNA topoisomerase I. In addition, we also report here a novel finding that purified recombinant six subunit ORC significantly stimulated the DNA synthesis on a supercoiled plasmid DNA template containing an autonomously replicating sequence, ARS1.     

Restriction cleavage map of mitochonrial DNA from the yeast Saccharomyces cerevisiae.

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb.