Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid pCW12, containing the ARG4gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid. Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome.     

Decade of genomics--methods for genome investigation in yeast Saccharomyces cerevisiae

The last ten years, since yeast Saccharomyces cerevisiae genome was sequenced, brought a big impact in genome-wide techniques. The tenth anniversary of genomic era provokes the following resume: a lot of new methods were invented. The yeast strains libraries carrying transposon insertions, gene deletions or tagged proteins have been created. Using them, the phenotypes of gene deletions, as well as the biological activity, cellular localization and possible modifications of their protein products were elucidated. SAGE analysis and DNA microarray experiments showed gene expression profiles and allowed to build interaction networks of gene regulation. The two dimensional gels, mass spectrometry, protein arrays and two-hybrid system carry information about protein interactions, modifications, and biochemical activities. All these methods permit to increase the number of genes with known cellular functions. Moreover, testing these techniques on yeast S. cerevisiae--a model eukaryotic organism--opened the door for their usage in all other species.     

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.     

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.     

Genetic requirements for the targeted integration of Agrobacterium T-DNA in Saccharomyces cerevisiae

Agrobacterium tumefaciens delivers transferred DNA (T-DNA) into cells of plants and yeast. In plants, the T-DNA integrates at random positions into the genome by non-homologous recombination (NHR), whereas in yeast the T-DNA preferably integrates by homologous recombination (HR). Here we show that T-DNA integration by HR in yeast requires the recombination/repair proteins Rad51 and Rad52, but not Rad50, Mre11, Xrs2, Yku70 and Lig4. In the HR events a remarkable shift from insertion-type events to replacement events was observed in rad50, mre11 and xrs2 mutants. Residual integration in the rad51 mutant occurred predominantly by HR, whereas in the rad52 mutant integration occurred exclusively by NHR. Previously, we found that T-DNA integration by NHR is abolished in a yku70 mutant. Thus, Rad52 and Yku70 are the key regulators of T-DNA integration, channeling integration into either the HR or NHR pathway.     

Features of integration of recombinant cosmids, containing Aspergillus terreus DNA, in the Saccharomyces cerevisiae genome

A genome clonotheque of 25-40 kb Sau3A fragments of Aspergillus terreus DNA was constructed in the episomal cosmid vector pES33 containing the ARG4 gene of yeast. 23 independently originated stable Arg+ transformants were selected after transformation of the cir0 yeast strain ESH-O with pooled cosmid molecules. Both genetic and Southern analysis showed that 39% of these stable transformants occurred due to recombination between DNA sequences from A. terreus and Saccharomyces cerevisiae chromosome XII which took place most likely in the regions of homology within the ribosomal clusters. The data present the first evidence of in vivo recombination between foreign sequences and their S. cerevisiae counterparts.     

Mitochondrial genome integrity mutations uncouple the yeast Saccharomyces cerevisiae ATP synthase

The mitochondrial ATP synthase is a molecular motor, which couples the flow of protons with phosphorylation of ADP. Rotation of the central stalk within the core of ATP synthase effects conformational changes in the active sites driving the synthesis of ATP. Mitochondrial genome integrity (mgi) mutations have been previously identified in the alpha-, beta-, and gamma-subunits of ATP synthase in yeast Kluyveromyces lactis and trypanosome Trypanosoma brucei. These mutations reverse the lethality of the loss of mitochondrial DNA in petite negative strains. Introduction of the homologous mutations in Saccharomyces cerevisiae results in yeast strains that lose mitochondrial DNA at a high rate and accompanied decreases in the coupling of the ATP synthase. The structure of yeast F1-ATPase reveals that the mgi residues cluster around the gamma-subunit and selectively around the collar region of F1. These results indicate that residues within the mgi complementation group are necessary for efficient coupling of ATP synthase, possibly acting as a support to fix the axis of rotation of the central stalk.     

Induction of genome instability by DNA damage in Saccharomyces cerevisiae

The accumulation of gross chromosomal rearrangements (GCRs) is a characteristic of many types of cancer cells, although it is unclear what defects cause these rearrangements and how the different types of GCRs observed are formed. In the present study, we have used a Saccharomyces cerevisiae system for measuring GCRs to analyze the ability of a variety of DNA damaging agents to induce GCRs. The two most potent inducers of GCRs observed were methyl methane sulfonate (MMS) and HO-endonuclease-induced double strand breaks (DSBs). Bleomycin, camptothecan and gamma-irradiation induced intermediate levels of GCRs and cisplatin induced very low levels of GCRs whereas N-methyl-NPRIME;-nitro-N-nitrosoguanidine (MNNG) and ethyl methane sulfonate (EMS) primarily induced base substitution mutations. MMS treatment primarily induced rearrangements in which the end of a chromosome was deleted and a new telomere was added (telomere additions) and also induced translocations. Consistent with this GCR spectrum, the formation of MMS-induced GCRs was primarily dependent on telomere maintenance functions and were completely eliminated in mutants that were defective for both telomere maintenance functions and non-homologous end joining (NHEJ). In contrast, HO-endonuclease DSBs induced mostly translocations and interstitial deletions whereas few telomere additions were observed. Genetic analysis indicated that HO DSB-induced GCRs were suppressed by a number of pathways including the DNA damage checkpoints, DSB repair pathways and NHEJ.     

Distribution and variability of trinucleotide repeats in the genome of the yeast Saccharomyces cerevisiae

We have examined the distribution of trinucleotide repeats in the yeast genome. Perfect and imperfect repeats, ranging from four to 130 triplets were recognized and the repartition of different triplet combinations was found to differ between Open Reading Frames and Intergenic Regions. Examination of different laboratory strains, revealed polymorphic size variations for all perfect repeats studied, compared to an absence of variation for the imperfect ones. Size variations were found discrete in the range of 6–18 triplets, each strain showing one allelic form for a given repeat array. The distribution and stability of trinucleotide repeats in the yeast genome resembles that of humans and may provide an experimental approach to study the mechanisms of their expansion.     

Ty1 integrase overexpression leads to integration of non-Ty1 DNA fragments into the genome of Saccharomyces cerevisiae

The integrase of the Saccharomyces cerevisiae retrotransposon Ty1 integrates Ty1 cDNA into genomic DNA likely via a transesterification reaction. Little is known about the mechanisms ensuring that integrase does not integrate non-Ty DNA fragments. In an effort to elucidate the conditions under which Ty1 integrase accepts non-Ty DNA as substrate, PCR fragments encompassing a selectable marker gene were transformed into yeast strains overexpressing Ty1 integrase. These fragments do not exhibit similarity to Ty1 cDNA except for the presence of the conserved terminal dinucleotide 5′-TG-CA-3′. The frequency of fragment insertion events increased upon integrase overexpression. Characterization of insertion events by genomic sequencing revealed that most insertion events exhibited clear hallmarks of integrase-mediated reactions, such as 5 bp target site duplication and target site preferences. Alteration of the terminal dinucleotide abolished the suitability of the PCR fragments to serve as substrates. We hypothesize that substrate specificity under normal conditions is mainly due to compartmentalization of integrase and Ty cDNA, which meet in virus-like particles. In contrast, recombinant integrase, which is not confined to virus-like particles, is able to accept non-Ty DNA, provided that it terminates in the proper dinucleotide sequence.     

3 micron DNA - an extrachromosomal ribosomal DNA in the yeast Saccharomyces cerevisiae

A new class of extrachromosomal DNA which consists predominantly of covalently closed molecules with lengths around 3 micron, has been detected in Saccharomyces cerevisiae strain 6-1G-P188 from the Peterhof collection. Restriction analysis of the 3 micron DNA as well as of recombinant plasmids carrying HindIII fragments of the 3 micron DNA permitted construction of a physical map of the new extrachromosomal DNA species, and detection of two types differing by one EcoRI restriction site. Molecular hybridization, as well as comparison of the restriction maps, revealed the complete structural identity of the 3 micron DNA with a chromosomal repetitive unit of rDNA containing the genes for 25 S, 18 S, 5.8 S and 5 S rRNAs.     

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.     

Iron-reductases in the yeast Saccharomyces cerevisiae

Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced in cells grown under iron-deficient conditions. At least two cytosolic iron-reducing enzymes having different substrate specificities could contribute to iron assimilation in vivo. One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses NADPH as electron donor and Fe(III)-EDTA as artificial electron acceptor. Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' NADH dehydrogenase, but not the siderophore ferrioxamine B. A plasma membrane-bound ferri-reductase system functioning with NADPH as electron donor and FMN as prosthetic group was purified 100-fold from isolated plasma membranes. This system may be involved in the reductive uptake of iron in vivo.     

Repair of double-strand breaks in plasmid DNA in the yeast Saccharomyces cerevisiae

Summary We studied the repair of double-strand breaks (DSB) in plasmid DNA introduced into haploid cells of the yeast Saccharomyces cerevisiae. The efficiency of repair was estimated from the frequency of transformation of the cells by an autonomously replicated linearized plasmid. The frequency of lithium transformation of Rad⁺ cells was increased greatly (by 1 order of magnitude and more) compared with that for circular DNA if the plasmid was initially linearized at the XhoI site within the LYS2 gene. This effect is due to recombinational repair of the plasmid DNA. Mutations rad52, rad53, rad54 and rad57 suppress the repair of DSB in plasmid DNA. The kinetics of DSB repair in plasmid DNA are biphasic: the first phase is completed within 1 h and the second within 14–18 h of incubating cells on selective medium.     

酿酒酵母基因组位置效应对外源基因表达的影响

外源基因元件和模块在底盘细胞中发挥特定功能是合成生物学研究的基本过程,而外源元件和模块在基因组中的位置对其功能的实现具有显著影响。为了系统、全面地表征酿酒酵母基因组位置效应对外源基因的表达影响,以绿色荧光蛋白为报告基因,通过双交换同源重组方法,对酿酒酵母单基因敲除库进行高通量转化,构建酿酒酵母基因组单位点荧光标记菌株库。结合流式细胞术和高通量测序技术对单位点荧光标记库菌株进行分析,构建高表达位点库和低表达位点库,共发现促进绿色荧光蛋白表达的位点428个,抑制绿色荧光蛋白表达的位点444个。通过分析高、低表达位点在酵母染色体上的分布,从全基因组尺度上对酿酒酵母基因组整合位置对基因表达的影响进行表征。本研究可为酿酒酵母基因组位置效应的分布规律和产生机理研究提供重要参考,对外源蛋白工业生产和合成生物学中的基因表达精细调控也具有重要的指导意义。     

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.     

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.     

Exopolyphosphatases of the yeast Saccharomyces cerevisiae

Separate compartments of the yeast cell possess their own exopolyphosphatases differing from each other in their properties and dependence on culture conditions. The low-molecular-mass exopolyphosphatases of the cytosol, cell envelope, and mitochondrial matrix are encoded by the PPX1 gene, while the high-molecular-mass exopolyphosphatase of the cytosol and those of the vacuoles, mitochondrial membranes, and nuclei are presumably encoded by their own genes. Based on recent works, a preliminary classification of the yeast exopolyphosphatases is proposed.