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Sample records for isogenic yeast dna

  1. Direct cloning of isogenic murine DNA in yeast and relevance of isogenicity for targeting in embryonic stem cells.

    PubMed

    Andréasson, Claes; Schick, Anna J; Pfeiffer, Susanne M; Sarov, Mihail; Stewart, Francis; Wurst, Wolfgang; Schick, Joel A

    2013-01-01

    Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors. PMID:24058528

  2. Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells

    PubMed Central

    Pfeiffer, Susanne M.; Sarov, Mihail; Stewart, Francis; Wurst, Wolfgang; Schick, Joel A.

    2013-01-01

    Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors. PMID:24058528

  3. Slow-growing cells within isogenic populations have increased RNA polymerase error rates and DNA damage.

    PubMed

    van Dijk, David; Dhar, Riddhiman; Missarova, Alsu M; Espinar, Lorena; Blevins, William R; Lehner, Ben; Carey, Lucas B

    2015-01-01

    Isogenic cells show a large degree of variability in growth rate, even when cultured in the same environment. Such cell-to-cell variability in growth can alter sensitivity to antibiotics, chemotherapy and environmental stress. To characterize transcriptional differences associated with this variability, we have developed a method--FitFlow--that enables the sorting of subpopulations by growth rate. The slow-growing subpopulation shows a transcriptional stress response, but, more surprisingly, these cells have reduced RNA polymerase fidelity and exhibit a DNA damage response. As DNA damage is often caused by oxidative stress, we test the addition of an antioxidant, and find that it reduces the size of the slow-growing population. More generally, we find a significantly altered transcriptome in the slow-growing subpopulation that only partially resembles that of cells growing slowly due to environmental and culture conditions. Slow-growing cells upregulate transposons and express more chromosomal, viral and plasmid-borne transcripts, and thus explore a larger genotypic--and so phenotypic--space. PMID:26268986

  4. Slow-growing cells within isogenic populations have increased RNA polymerase error rates and DNA damage

    PubMed Central

    van Dijk, David; Dhar, Riddhiman; Missarova, Alsu M.; Espinar, Lorena; Blevins, William R.; Lehner, Ben; Carey, Lucas B.

    2015-01-01

    Isogenic cells show a large degree of variability in growth rate, even when cultured in the same environment. Such cell-to-cell variability in growth can alter sensitivity to antibiotics, chemotherapy and environmental stress. To characterize transcriptional differences associated with this variability, we have developed a method—FitFlow—that enables the sorting of subpopulations by growth rate. The slow-growing subpopulation shows a transcriptional stress response, but, more surprisingly, these cells have reduced RNA polymerase fidelity and exhibit a DNA damage response. As DNA damage is often caused by oxidative stress, we test the addition of an antioxidant, and find that it reduces the size of the slow-growing population. More generally, we find a significantly altered transcriptome in the slow-growing subpopulation that only partially resembles that of cells growing slowly due to environmental and culture conditions. Slow-growing cells upregulate transposons and express more chromosomal, viral and plasmid-borne transcripts, and thus explore a larger genotypic—and so phenotypic — space. PMID:26268986

  5. Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines

    PubMed Central

    Yamamoto, Kimiyo N.; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P.; Witt, Kristine L.; Tice, Raymond R.

    2012-01-01

    Included among the quantitative high throughput screens (qHTS) conducted in support of the U.S. Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in 7 isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis. PMID:21538559

  6. Assessment of Chromosomal DNA Fragmentation by Quinolones in an Isogenic Collection of Escherichia coli with Defined Resistance Mechanisms.

    PubMed

    Rodríguez-Martínez, José-Manuel; Santiso, Rebeca; Machuca, Jesús; Bou, Germán; Pascual, Álvaro; Fernández, José Luis

    2016-07-01

    The aim of this study was to investigate the potential usefulness of DNA fragmentation as a quick and simple procedure for detecting resistance to fluoroquinolones (FQ) in isogenic Escherichia coli strains harboring defined and multiple quinolone resistance mechanisms, including low-level quinolone resistance (LLQR) phenotypes. DNA fragmentation assay (Micromax(®)) was evaluated for detecting resistance to FQ in 71 isogenic strains of E. coli harboring specific quinolone resistance mechanisms frequently found in clinical isolates. These isogenic strains represent a consistent and reliable model of increasing minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP), ranging from 0.004 to 16 mg/L. According to CLSI criteria, the assay correctly identified all CIP-resistant strains (MIC ≥4 mg/L). As regards susceptible strains, 96% of bacterial strains were correctly assigned as susceptible to CIP. Moreover, the procedure enabled LLQR phenotypes to be efficiently identified; this subset may show different levels of DNA damage depending on the strain, even with similar MIC. Interestingly, despite increasing the dose according to the MIC, a lower response to quinolones occurs in strains with higher MIC values. This is a simple, rapid, and reliable test for evaluating susceptibility to FQ of E. coli, including the detection of strains harboring LLQR mechanisms. PMID:26890225

  7. Size and Structure of Yeast Chromosomal DNA

    PubMed Central

    Petes, Thomas D.; Byers, Breck; Fangman, Walton L.

    1973-01-01

    Electron microscopic analysis indicates that yeast nuclear DNA can be isolated as linear molecules ranging in size from 50 μm (1.2 × 108 daltons) to 355 μm (8.4 × 108 daltons). Analysis indicates the data is consistent with the hypothesis that each yeast chromosome contains a single, linear DNA duplex. Mitochondrial DNA molecules have a contour length of 21 ± 2 μm and are mostly linear. Images PMID:4594033

  8. Visualization of yeast chromosomal DNA

    NASA Technical Reports Server (NTRS)

    Lubega, Seth

    1990-01-01

    The DNA molecule is the most significant life molecule since it codes the blue print for other structural and functional molecules of all living organisms. Agarose gel electrophoresis is now being widely used to separate DNA of virus, bacteria, and lower eukaryotes. The task was undertaken of reviewing the existing methods of DNA fractionation and microscopic visualization of individual chromosonal DNA molecules by gel electrophoresis as a basis for a proposed study to investigate the feasibility of separating DNA molecules in free fluids as an alternative to gel electrophoresis. Various techniques were studied. On the molecular level, agarose gel electrophoresis is being widely used to separate chromosomal DNA according to molecular weight. Carl and Olson separate and characterized the entire karyotype of a lab strain of Saccharomyces cerevisiae. Smith et al. and Schwartz and Koval independently reported the visualization of individual DNA molecules migrating through agarose gel matrix during electrophoresis. The techniques used by these researchers are being reviewed in the lab as a basis for the proposed studies.

  9. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome

    PubMed Central

    DeKelver, Russell C.; Choi, Vivian M.; Moehle, Erica A.; Paschon, David E.; Hockemeyer, Dirk; Meijsing, Sebastiaan H.; Sancak, Yasemin; Cui, Xiaoxia; Steine, Eveline J.; Miller, Jeffrey C.; Tam, Phillip; Bartsevich, Victor V.; Meng, Xiangdong; Rupniewski, Igor; Gopalan, Sunita M.; Sun, Helena C.; Pitz, Kathleen J.; Rock, Jeremy M.; Zhang, Lei; Davis, Gregory D.; Rebar, Edward J.; Cheeseman, Iain M.; Yamamoto, Keith R.; Sabatini, David M.; Jaenisch, Rudolf; Gregory, Philip D.; Urnov, Fyodor D.

    2010-01-01

    Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a “safe harbor” locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells. PMID:20508142

  10. Extrachromosomal circular DNA is common in yeast

    PubMed Central

    Møller, Henrik D.; Parsons, Lance; Jørgensen, Tue S.; Botstein, David; Regenberg, Birgitte

    2015-01-01

    Examples of extrachromosomal circular DNAs (eccDNAs) are found in many organisms, but their impact on genetic variation at the genome scale has not been investigated. We mapped 1,756 eccDNAs in the Saccharomyces cerevisiae genome using Circle-Seq, a highly sensitive eccDNA purification method. Yeast eccDNAs ranged from an arbitrary lower limit of 1 kb up to 38 kb and covered 23% of the genome, representing thousands of genes. EccDNA arose both from genomic regions with repetitive sequences ≥15 bases long and from regions with short or no repetitive sequences. Some eccDNAs were identified in several yeast populations. These eccDNAs contained ribosomal genes, transposon remnants, and tandemly repeated genes (HXT6/7, ENA1/2/5, and CUP1-1/-2) that were generally enriched on eccDNAs. EccDNAs seemed to be replicated and 80% contained consensus sequences for autonomous replication origins that could explain their maintenance. Our data suggest that eccDNAs are common in S. cerevisiae, where they might contribute substantially to genetic variation and evolution. PMID:26038577

  11. Mechanism for priming DNA synthesis by yeast DNA Polymerase α

    PubMed Central

    Perera, Rajika L; Torella, Rubben; Klinge, Sebastian; Kilkenny, Mairi L; Maman, Joseph D; Pellegrini, Luca

    2013-01-01

    The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI: http://dx.doi.org/10.7554/eLife.00482.001 PMID:23599895

  12. Recent advances in yeast molecular biology: recombinant DNA. [Lead abstract

    SciTech Connect

    Not Available

    1982-09-01

    Separate abstracts were prepared for the 25 papers presented at a workshop focusing on chromosomal structure, gene regulation, recombination, DNA repair, and cell type control, that have been obtained by experimental approaches incorporating the new technologies of yeast DNA transformation, molecular cloning, and DNA sequence analysis. (KRM)

  13. Isolation of Circular DNA from a Mitochondrial Fraction from Yeast

    PubMed Central

    Clark-Walker, G. D.

    1972-01-01

    Breakage and fractionation of respiratory competent yeast in the presence of ethidium bromide, and subsequent centrifugation of a detergent lysate of the mitochondrial fraction by the dye-buoyant-density technique, results in the isolation of closed-circular DNA. After removal of bound dye, this DNA has two components when analyzed by equilibrium buoyant density in the analytical ultracentrifuge. A minor component has a buoyant density of 1.684 g/cm3, which is characteristic of mitochondrial DNA, but the major component has a buoyant density of 1.701 g/cm3. This species of DNA is also present in yeast that have been mutagenized to respiratory deficiency in the presence of the highest concentration of ethidium bromide compatible with cell growth. The closed-circular DNA of buoyant density 1.701 g/cm3, and free of linear DNA, is associated with the sole particulate band obtained on sucrose gradient centrifugation of a mitochondrial preparation from respiratory-deficient cells. Two particulate bands are obtained on sucrose gradient centrifugation of a mitochondrial preparation from respiratory-competent cells, the upper band containing DNA of buoyant density 1.701 g/cm3 and the lower band DNA of buoyant density 1.684 g/cm3. The suggestion is advanced, in view of the reputed sedimentation behaviour of yeast peroxisomes, that the closed-circular DNA of buoyant density 1.701 g/cm3 may be located in peroxisomes. Images PMID:4551142

  14. Modeling the Control of DNA Replication in Fission Yeast

    NASA Astrophysics Data System (ADS)

    Novak, Bela; Tyson, John J.

    1997-08-01

    A central event in the eukaryotic cell cycle is the decision to commence DNA replication (S phase). Strict controls normally operate to prevent repeated rounds of DNA replication without intervening mitoses (``endoreplication'') or initiation of mitosis before DNA is fully replicated (``mitotic catastrophe''). Some of the genetic interactions involved in these controls have recently been identified in yeast. From this evidence we propose a molecular mechanism of ``Start'' control in Schizosaccharomyces pombe. Using established principles of biochemical kinetics, we compare the properties of this model in detail with the observed behavior of various mutant strains of fission yeast: wee1- (size control at Start), cdc13Δ and rum1OP (endoreplication), and wee1- rum1Δ (rapid division cycles of diminishing cell size). We discuss essential features of the mechanism that are responsible for characteristic properties of Start control in fission yeast, to expose our proposal to crucial experimental tests.

  15. DNA sequence of the yeast transketolase gene.

    PubMed

    Fletcher, T S; Kwee, I L; Nakada, T; Largman, C; Martin, B M

    1992-02-18

    Transketolase (EC 2.2.1.1) is the enzyme that, together with aldolase, forms a reversible link between the glycolytic and pentose phosphate pathways. We have cloned and sequenced the transketolase gene from yeast (Saccharomyces cerevisiae). This is the first transketolase gene of the pentose phosphate shunt to be sequenced from any source. The molecular mass of the proposed translated protein is 73,976 daltons, in good agreement with the observed molecular mass of about 75,000 daltons. The 5'-nontranslated region of the gene is similar to other yeast genes. There is no evidence of 5'-splice junctions or branch points in the sequence. The 3'-nontranslated region contains the polyadenylation signal (AATAAA), 80 base pairs downstream from the termination codon. A high degree of homology is found between yeast transketolase and dihydroxyacetone synthase (formaldehyde transketolase) from the yeast Hansenula polymorpha. The overall sequence identity between these two proteins is 37%, with four regions of much greater similarity. The regions from amino acid residues 98-131, 157-182, 410-433, and 474-489 have sequence identities of 74%, 66%, 83%, and 82%, respectively. One of these regions (157-182) includes a possible thiamin pyrophosphate (TPP) binding domain, and another (410-433) may contain the catalytic domain. PMID:1737042

  16. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  17. The Yeast Copper Response Is Regulated by DNA Damage

    PubMed Central

    Dong, Kangzhen; Addinall, Stephen G.; Lydall, David

    2013-01-01

    Copper is an essential but potentially toxic redox-active metal, so the levels and distribution of this metal are carefully regulated to ensure that it binds to the correct proteins. Previous studies of copper-dependent transcription in the yeast Saccharomyces cerevisiae have focused on the response of genes to changes in the exogenous levels of copper. We now report that yeast copper genes are regulated in response to the DNA-damaging agents methyl methanesulfonate (MMS) and hydroxyurea by a mechanism(s) that requires the copper-responsive transcription factors Mac1 and AceI, copper superoxide dismutase (Sod1) activity, and the Rad53 checkpoint kinase. Furthermore, in copper-starved yeast, the response of the Rad53 pathway to MMS is compromised due to a loss of Sod1 activity, consistent with the model that yeast imports copper to ensure Sod1 activity and Rad53 signaling. Crucially, the Mac1 transcription factor undergoes changes in its redox state in response to changing levels of copper or MMS. This study has therefore identified a novel regulatory relationship between cellular redox, copper homeostasis, and the DNA damage response in yeast. PMID:23959798

  18. Mitochondrial DNA repairs double-strand breaks in yeast chromosomes.

    PubMed

    Ricchetti, M; Fairhead, C; Dujon, B

    1999-11-01

    The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process. PMID:10573425

  19. DNA Compaction by Yeast Mitochondrial Protein ABF2p

    SciTech Connect

    Friddle, R W; Klare, J E; Noy, A; Corzett, M; Balhorn, R; Baskin, R J; Martin, S S; Baldwin, E P

    2003-05-09

    We used high resolution Atomic Force Microscopy (AFM) to image compaction of linear and circular DNA by the yeast mitochondrial protein ABF2p , which plays a major role in maintaining mitochondrial DNA. AFM images show that protein binding induces drastic bends in the DNA backbone for both linear and circular DNA. At high concentration of ABF2p DNA collapses into a tight globular structure. We quantified the compaction of linear DNA by measuring the end-to-end distance of the DNA molecule at increasing concentrations of ABF2p. We also derived a polymer statistical mechanics model that gives quantitative description of compaction observed in our experiments. This model shows that a number of sharp bends in the DNA backbone is often sufficient to cause DNA compaction. Comparison of our model with the experimental data showed excellent quantitative correlation and allowed us to determine binding characteristics for ABF2. Our studies indicate that ABF2 compacts DNA through a novel mechanism that involves bending of DNA backbone. We discuss the implications of such a mechanism for mitochondrial DNA maintenance.

  20. Mitochondrial DNA size diversity in the Dekkera/Brettanomyces yeasts.

    PubMed

    McArthur, C R; Clark-Walker, G D

    1983-03-01

    Restriction endonuclease digestion of mitocondrial DNAs from the nine Dekkera/Brettanomyces yeasts have revealed that three separate pairs of species, namely D. bruxellensis/B. lambicus; B. abstinens/B. custersii and B. anomalus/B. clausenii have identical genomes. This observation suggests that such analysis of mtDNA could be an important procedure for yeast taxonomy. Sizes of mtDNAs showed a graded range from the 28 kbp molecule in B. custersianus to the 100 kbp molecule in B. custersii. Furthermore, although the mtDNAs from D. intermedia (72 kbp) and D. bruxellensis (82 kbp) differ in size by 10 kbp the restriction enzyme fragmentation patterns are generally similar. The differences are reminiscent of mtDNA polymorphisms found in strains of Saccharomyces cervisiae which result from insertions or deletions, chiefly within genic sequences. By analogy, the two Dekkera species may, on further analysis, be revealed as variants of a single species. PMID:24173115

  1. Structural Basis of High-Fidelity DNA Synthesis by Yeast DNA Polymerase δ

    SciTech Connect

    Swan, M.; Johnson, R; Prakash, L; Prakash, S; Aggarwal, A

    2009-01-01

    DNA polymerase ? (Pol ?) has a crucial role in eukaryotic replication. Now the crystal structure of the yeast DNA Pol ? catalytic subunit in complex with template primer and incoming nucleotide is presented at 2.0-A resolution, providing insight into its high fidelity and a framework to understand the effects of mutations involved in tumorigenesis.

  2. Single-stranded-DNA-binding protein-dependent DNA unwinding of the yeast ARS1 region.

    PubMed Central

    Matsumoto, K; Ishimi, Y

    1994-01-01

    DNA unwinding of autonomously replicating sequence 1 (ARS1) from the yeast Saccharomyces cerevisiae was investigated. When a negatively supercoiled plasmid DNA containing ARS1 was digested with single-strand-specific mung bean nuclease, a discrete region in the vector DNA was preferentially digested. The regions containing the core consensus A domain and the 3'-flanking B domain of ARS1 were weakly digested. When the DNA was incubated with the multisubunit single-stranded DNA-binding protein (SSB, also called RPA [replication protein A]) from human and yeast cells prior to mung bean nuclease digestion, the cleavage in the A and B domains was greatly increased. Furthermore, a region corresponding to the 5'-flanking C domain of ARS1 was digested. These results indicate that three domains of ARS1, each of which is important for replication in yeast cells, closely correspond to the regions where the DNA duplex is easily unwound by torsional stress. SSB may stimulate the unwinding of the ARS1 region by its preferential binding to the destabilized three domains. Mung bean nuclease digestion of the substitution mutants with mutations of ARS1 (Y. Marahrens and B. Stillman, Science 255:817-823, 1992) revealed that the sequences in the B2 and A elements are responsible for the unwinding of the B domain and the region containing the A domain, respectively. Images PMID:8007967

  3. Quantitative PCR for detection of DNA damage in mitochondrial DNA of the fission yeast Schizosaccharomyces pombe.

    PubMed

    Senoo, Takanori; Yamanaka, Mayumi; Nakamura, Atori; Terashita, Tomoki; Kawano, Shinji; Ikeda, Shogo

    2016-08-01

    Quantitative polymerase chain reaction (QPCR) has been employed to detect DNA damage and repair in mitochondrial DNA (mtDNA) of human and several model organisms. The assay also permits the quantitation of relative mtDNA copy number in cells. Here, we developed the QPCR assay primers and reaction conditions for the fission yeast Schizosaccharomyces pombe, an important model of eukaryote biology, not previously described. Under these conditions, long targets (approximately 10kb) in mtDNA were quantitatively amplified using 0.1ng of crude DNA templates without isolation of mitochondria and mtDNA. Quantitative detection of oxidative DNA damage in mtDNA was illustrated by using a DNA template irradiated with UVA in the presence of riboflavin. The damage to mtDNA in S. pombe cells treated with hydrogen peroxide and paraquat was also quantitatively measured. Finally, we found that mtDNA copy number in S. pombe cells increased after transition into a stationary phase and that the damage to mtDNA due to endogenous cellular processes accumulated during chronological aging. PMID:27236021

  4. Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

    PubMed Central

    Makarova, Alena V.; Grabow, Corinn; Gening, Leonid V.; Tarantul, Vyacheslav Z.; Tahirov, Tahir H.; Bessho, Tadayoshi; Pavlov, Youri I.

    2011-01-01

    Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts. PMID:21304950

  5. Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene.

    PubMed Central

    Thrash, C; Bankier, A T; Barrell, B G; Sternglanz, R

    1985-01-01

    The structural gene for yeast DNA topoisomerase I (TOP1) has been cloned from two yeast genomic plasmid banks. Integration of a plasmid carrying the gene into the chromosome and subsequent genetic mapping shows that TOP1 is identical to the gene previously called MAK1. Seven top1 (mak1) mutants including gene disruptions are viable, demonstrating that DNA topoisomerase I is not essential for viability in yeast. A 3787-base-pair DNA fragment including the gene has been sequenced. The protein predicted from the DNA sequence has 769 amino acids and a molecular weight of 90,020. Images PMID:2989818

  6. Structural basis of high-fidelity DNA synthesis by yeast DNA polymerase [delta

    SciTech Connect

    Swan, Michael K.; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2009-09-25

    DNA polymerase {delta} (Pol {delta}) is a high-fidelity polymerase that has a central role in replication from yeast to humans. We present the crystal structure of the catalytic subunit of yeast Pol {delta} in ternary complex with a template primer and an incoming nucleotide. The structure, determined at 2.0-{angstrom} resolution, catches the enzyme in the act of replication, revealing how the polymerase and exonuclease domains are juxtaposed relative to each other and how a correct nucleotide is selected and incorporated. The structure also reveals the 'sensing' interactions near the primer terminus, which signal a switch from the polymerizing to the editing mode. Taken together, the structure provides a chemical basis for the bulk of DNA synthesis in eukaryotic cells and a framework for understanding the effects of cancer-causing mutations in Pol {delta}.

  7. Repair of UV damaged DNA, genes and proteins of yeast and human

    SciTech Connect

    Prakash, L.

    1991-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, as well as studying the human homologs of yeast excision repair and postreplication repair proteins. In addition to its single-stranded DNA-dependent A TPase and DNA helicase activities, we have found that RAD3 protein also possesses DNA-RNA helicase activity, and that like RAD3, the Schizosaccharomyces pombe RAD3 homolog, rhp3{sup +}, is an essential gene. We have overexpressed the human RAD3 homolog, ERCC2, in yeast to facilitate its purification. The RAD10 protein was purified to homogeneity and shown to bind DNA. ERCC3y, the yeast homolog of the human ERCC-3/XP-B gene, has been sequenced and shown to be essential for viability. The Drosophila and human homologs of RAD6, required for postreplication repair and UV induced mutagenesis, were shown to complement the rad6 {Delta} mutation of yeast. Since defective DNA repair and enhanced neoplasia characterize several human genetic diseases, and repair proteins are highly conserved between yeast and man, a thorough understanding of the molecular mechanisms of DNA repir in yeast should provide a better understanding of the causes of carcinogenesis.

  8. Rif1: A Conserved Regulator of DNA Replication and Repair Hijacked by Telomeres in Yeasts

    PubMed Central

    Mattarocci, Stefano; Hafner, Lukas; Lezaja, Aleksandra; Shyian, Maksym; Shore, David

    2016-01-01

    Rap1-interacting factor 1 (Rif1) was originally identified in the budding yeast Saccharomyces cerevisiae as a telomere-binding protein that negatively regulates telomerase-mediated telomere elongation. Although this function is conserved in the distantly related fission yeast Schizosaccharomyces pombe, recent studies, both in yeasts and in metazoans, reveal that Rif1 also functions more globally, both in the temporal control of DNA replication and in DNA repair. Rif1 proteins are large and characterized by N-terminal HEAT repeats, predicted to form an elongated alpha-helical structure. In addition, all Rif1 homologs contain two short motifs, abbreviated RVxF/SILK, that are implicated in recruitment of the PP1 (yeast Glc7) phosphatase. In yeasts the RVxF/SILK domains have been shown to play a role in control of DNA replication initiation, at least in part through targeted de-phosphorylation of proteins in the pre-Replication Complex. In human cells Rif1 is recruited to DNA double-strand breaks through an interaction with 53BP1 where it counteracts DNA resection, thus promoting repair by non-homologous end-joining. This function requires the N-terminal HEAT repeat-containing domain. Interestingly, this domain is also implicated in DNA end protection at un-capped telomeres in yeast. We conclude by discussing the deployment of Rif1 at telomeres in yeasts from both an evolutionary perspective and in light of its recently discovered global functions. PMID:27066066

  9. Extraction of genomic DNA from yeasts for PCR-based applications.

    PubMed

    Lõoke, Marko; Kristjuhan, Kersti; Kristjuhan, Arnold

    2011-05-01

    We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp. PMID:21548894

  10. Amplification of a Zygosaccharomyces bailii DNA Segment in Wine Yeast Genomes by Extrachromosomal Circular DNA Formation

    PubMed Central

    Galeote, Virginie; Bigey, Frédéric; Beyne, Emmanuelle; Novo, Maite; Legras, Jean-Luc; Casaregola, Serge; Dequin, Sylvie

    2011-01-01

    We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT) events in the genome of Saccharomyces cerevisiae strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from Zygosaccharomyces bailii, in the genome of S. cerevisiae strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28 S. cerevisiae strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in S. cerevisiae, strongly suggest that an extrachromosomal circular DNA (eccDNA) molecule serves as an intermediate in the amplification of the Z. bailii region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in S. cerevisiae represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations. PMID:21423766

  11. Repair of uv damaged DNA: Genes and proteins of yeast and human

    SciTech Connect

    Prakash, L.

    1992-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, and to study the human homologs of yeast excision repair and postreplication repair proteins progress is described.

  12. Gene sequence analyses and other DNA-based methods for yeast species recognition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA sequence analyses, as well as other DNA-based methodologies, have transformed the way in which yeasts are identified. The focus of this chapter will be on the resolution of species using various types of DNA comparisons. In other chapters in this book, Rozpedowska, Piškur and Wolfe discuss mul...

  13. Mitotic entry in the presence of DNA damage is a widespread property of aneuploidy in yeast

    PubMed Central

    Blank, Heidi M.; Sheltzer, Jason M.; Meehl, Colleen M.; Amon, Angelika

    2015-01-01

    Genetic instability is a hallmark of aneuploidy in budding and fission yeast. All aneuploid yeast strains analyzed to date harbor elevated levels of Rad52-GFP foci, a sign of DNA damage. Here we investigate how continuously elevated levels of DNA damage affect aneuploid cells. We show that Rad52-GFP foci form during S phase, consistent with the observation that DNA replication initiation and elongation are impaired in some aneuploid yeast strains. We furthermore find that although DNA damage is low in aneuploid cells, it nevertheless has dramatic consequences. Many aneuploid yeast strains adapt to DNA damage and undergo mitosis despite the presence of unrepaired DNA leading to cell death. Wild-type cells exposed to low levels of DNA damage exhibit a similar phenotype, indicating that adaptation to low levels of unrepaired DNA is a general property of the cell's response to DNA damage. Our results indicate that by causing low levels of DNA damage, whole-chromosome aneuploidies lead to DNA breaks that persist into mitosis. Such breaks provide the substrate for translocations and deletions that are a hallmark of cancer. PMID:25694455

  14. RNA∶DNA hybrids initiate quasi-palindrome-associated mutations in highly transcribed yeast DNA.

    PubMed

    Kim, Nayun; Cho, Jang-Eun; Li, Yue C; Jinks-Robertson, Sue

    2013-11-01

    RNase H enzymes promote genetic stability by degrading aberrant RNA:DNA hybrids and by removing ribonucleotide monophosphates (rNMPs) that are present in duplex DNA. Here, we report that loss of RNase H2 in yeast is associated with mutations that extend identity between the arms of imperfect inverted repeats (quasi-palindromes or QPs), a mutation type generally attributed to a template switch during DNA synthesis. QP events were detected using frameshift-reversion assays and were only observed under conditions of high transcription. In striking contrast to transcription-associated short deletions that also are detected by these assays, QP events do not require Top1 activity. QP mutation rates are strongly affected by the direction of DNA replication and, in contrast to their elevation in the absence of RNase H2, are reduced when RNase H1 is additionally eliminated. Finally, transcription-associated QP events are limited by components of the nucleotide excision repair pathway and are promoted by translesion synthesis DNA polymerases. We suggest that QP mutations reflect either a transcription-associated perturbation of Okazaki-fragment processing, or the use of a nascent transcript to resume replication following a transcription-replication conflict. PMID:24244191

  15. DNA synthesis in yeast cell-free extracts dependent on recombinant DNA plasmids purified from Escherichia coli.

    PubMed Central

    Jong, A Y; Scott, J F

    1985-01-01

    In our attempts to establish a cell-free DNA replication system for the yeast Saccharomyces cerevisiae, we have observed that recombinant DNA plasmids purified from Escherichia coli by a common procedure (lysozyme-detergent lysis and equilibrium banding in cesium chloride ethidium bromide gradients) often serve as templates for DNA synthesis by elongation enzymes. The templates could be elongated equally well by enzymes present in the yeast cell-free extracts, by the large proteolytic fragment of E. coli DNA polymerase I or by T4 DNA polymerase. The template activity of the purified plasmids was dependent on the presence of heterologous DNA segments in the bacterial vectors. The template activity could be diminished by treatment with alkali. We propose that the ability of recombinant plasmids isolated from bacterial hosts to serve as elongation templates may lead to erroneous conclusions when these plasmids are used as templates for in vitro replication or transcription reactions. Images PMID:3889851

  16. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  17. Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector.

    PubMed

    Hou, Zheng; Zhou, Zheng; Wang, Zonglin; Xiao, Gengfu

    2016-04-01

    Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named pGF (plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid pCC1BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome (YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction (PCR) from the plasmid pBS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination (TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using pGF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction. PMID:27113243

  18. A single protocol for extraction of gDNA from bacteria and yeast.

    PubMed

    Vingataramin, Laurie; Frost, Eric H

    2015-03-01

    Guanidine thiocyanate breakage of microorganisms has been the standard initial step in genomic DNA (gDNA) extraction of microbial DNA for two decades, despite the requirement for pretreatments to extract DNA from microorganisms other than Gram-negative bacteria. We report a quick and low-cost gDNA extraction protocol called EtNa that is efficient for bacteria and yeast over a broad range of concentrations. EtNa is based on a hot alkaline ethanol lysis. The solution can be immediately centrifuged to yield a crude gDNA extract suitable for PCR, or it can be directly applied to a silica column for purification. PMID:25757544

  19. Structural Basis for Error-free Replication of Oxidatively Damaged DNA by Yeast DNA Polymerase eta

    SciTech Connect

    T Silverstein; R Jain; R Johnson; L Prakash; S Prakash; A Aggarwal

    2011-12-31

    7,8-dihydro-8-oxoguanine (8-oxoG) adducts are formed frequently by the attack of oxygen-free radicals on DNA. They are among the most mutagenic lesions in cells because of their dual coding potential, where, in addition to normal base-pairing of 8-oxoG(anti) with dCTP, 8-oxoG in the syn conformation can base pair with dATP, causing G to T transversions. We provide here for the first time a structural basis for the error-free replication of 8-oxoG lesions by yeast DNA polymerase {eta} (Pol{eta}). We show that the open active site cleft of Pol{eta} can accommodate an 8-oxoG lesion in the anti conformation with only minimal changes to the polymerase and the bound DNA: at both the insertion and post-insertion steps of lesion bypass. Importantly, the active site geometry remains the same as in the undamaged complex and provides a basis for the ability of Pol to prevent the mutagenic replication of 8-oxoG lesions in cells.

  20. Relationships between rDNA, Nop1 and Sir complex in biotechnologically relevant distillery yeasts.

    PubMed

    Adamczyk, Jagoda; Deregowska, Anna; Potocki, Leszek; Kuna, Ewelina; Kaplan, Jakub; Pabian, Sylwia; Kwiatkowska, Aleksandra; Lewinska, Anna; Wnuk, Maciej

    2016-09-01

    Distillery yeasts are poorly characterized physiological group among the Saccharomyces sensu stricto complex. As industrial yeasts are under constant environmental stress during fermentation processes and the nucleolus is a stress sensor, in the present study, nucleolus-related parameters were evaluated in 22 commercially available distillery yeast strains. Distillery yeasts were found to be a heterogeneous group with a variable content and length of rDNA and degree of nucleolus fragmentation. The levels of rDNA were negatively correlated with Nop1 (r = -0.59, p = 0.0038). Moreover, the protein levels of Sir transcriptional silencing complex and longevity regulators, namely Sir1, Sir2, Sir3 and Fob1, were studied and negative correlations between Sir2 and Nop1 (r = -0.45, p = 0.0332), and between Sir2 and Fob1 (r = -0.49, p = 0.0211) were revealed. In general, S. paradoxus group of distillery yeasts with higher rDNA pools and Sir2 level than S. bayanus group was found to be more tolerant to fermentation-associated stress stimuli, namely mild cold/heat stresses and KCl treatment. We postulate that rDNA state may be considered as a novel factor that may modulate a biotechnological process. PMID:27329282

  1. The yeast telomere length regulator TEL2 encodes a protein that binds to telomeric DNA.

    PubMed Central

    Kota, R S; Runge, K W

    1998-01-01

    TEL2 is required for telomere length regulation and viability in Saccharomyces cerevisiae. To investigate the mechanism by which Tel2p regulates telomere length, the majority (65%) of the TEL2 ORF was fused to the 3'-end of the gene for maltose binding protein, expressed in bacteria and the purified protein used in DNA binding studies. Rap1p, the major yeast telomere binding protein, recognizes a 13 bp duplex site 5'-GGTGTGTGGGTGT-3' in yeast telomeric DNA with high affinity. Gel shift experiments revealed that the MBP-Tel2p fusion binds the double-stranded yeast telomeric Rap1p site in a sequence-specific manner. Analysis of mutated sites showed that MBP-Tel2p could bind 5'-GTGTGTGG-3' within this 13 bp site. Methylation interference analysis revealed that Tel2p contacts the 5'-terminal guanine in the major groove. MBP-Tel2p did not bind duplex telomeric DNA repeats from vertebrates, Tetrahymena or Oxytricha. These results suggest that Tel2p is a DNA binding protein that recognizes yeast telomeric DNA. PMID:9490802

  2. DNA polymerase γ and disease: what we have learned from yeast

    PubMed Central

    Lodi, Tiziana; Dallabona, Cristina; Nolli, Cecilia; Goffrini, Paola; Donnini, Claudia; Baruffini, Enrico

    2015-01-01

    Mip1 is the Saccharomyces cerevisiae DNA polymerase γ (Pol γ), which is responsible for the replication of mitochondrial DNA (mtDNA). It belongs to the family A of the DNA polymerases and it is orthologs to human POLGA. In humans, mutations in POLG(1) cause many mitochondrial pathologies, such as progressive external ophthalmoplegia (PEO), Alpers' syndrome, and ataxia-neuropathy syndrome, all of which present instability of mtDNA, which results in impaired mitochondrial function in several tissues with variable degrees of severity. In this review, we summarize the genetic and biochemical knowledge published on yeast mitochondrial DNA polymerase from 1989, when the MIP1 gene was first cloned, up until now. The role of yeast is particularly emphasized in (i) validating the pathological mutations found in human POLG and modeled in MIP1, (ii) determining the molecular defects caused by these mutations and (iii) finding the correlation between mutations/polymorphisms in POLGA and mtDNA toxicity induced by specific drugs. We also describe recent findings regarding the discovery of molecules able to rescue the phenotypic defects caused by pathological mutations in Mip1, and the construction of a model system in which the human Pol γ holoenzyme is expressed in yeast and complements the loss of Mip1. PMID:25852747

  3. The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA.

    PubMed

    Ramachandran, Aparna; Nandakumar, Divya; Deshpande, Aishwarya P; Lucas, Thomas P; R-Bhojappa, Ramanagouda; Tang, Guo-Qing; Raney, Kevin; Yin, Y Whitney; Patel, Smita S

    2016-08-01

    Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA. PMID:27311715

  4. Evidence that a single DNA ligase is involved in replication and recombination in yeast.

    PubMed Central

    Fabre, F; Roman, H

    1979-01-01

    The possible existence in yeast of different nuclear DNA ligase enzymes led us to ask whether induced recombination (gene conversion) involves the same ligase as that involved in DNA replication. The conditional cdc9 mutant is known to be defective, under restrictive conditions, in the rejoining of Okazaki fragments. We show here that under the same conditions, x-ray-induced convertants within the cdc9 locus are produced with kinetics indicating that most, if not all, of the conversion events require the participation of the cdc9-controlled ligase. Thus, the same DNA ligase is involved in DNA replication and in induced gene conversion. PMID:388446

  5. Heavy ion induced DNA-DSB in yeast and mammalian cells

    NASA Technical Reports Server (NTRS)

    Loebrich, M.; Ikpeme, S.; Kiefer, J.

    1994-01-01

    Molecular changes at the DNA are assumed to be the main cause for radiation effects in a number of organisms. During the course of the last decades techniques have been developed for measuring DNA double-strand breaks (dsb), generally assumed to be the most critical DNA lesions. The outcome of all those different approaches portrays a collection of data useful for a theoretical description of radiation action mechanisms. However, in the case of heavy ion induced DNA dsb the picture is not quite clear yet and further projects and strategies have to be developed. The biological systems studied in our group are yeast and mammalian cells. While in the case of yeast cells technical and methodical reasons highlight these organisms mammalian cells reach greater importance when dsb repair studies are performed. In both types of organisms the technique of pulsed-field gel electrophoresis (PFGE) is applied, although with different modifications and evaluation procedures mainly due to the different genome sizes.

  6. GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA

    PubMed Central

    Blount, Benjamin A.; Driessen, Maureen R. M.; Ellis, Tom

    2016-01-01

    Existing yeast genomic DNA extraction methods are not ideally suited to extensive screening of colonies by PCR, due to being too lengthy, too laborious or yielding poor quality DNA and inconsistent results. We developed the GC prep method as a solution to this problem. Yeast cells from colonies or liquid cultures are lysed by vortex mixing with glass beads and then boiled in the presence of a metal chelating resin. In around 12 minutes, multiple samples can be processed to extract high yields of genomic DNA. These preparations perform as effectively in PCR screening as DNA purified by organic solvent methods, are stable for up to 1 year at room temperature and can be used as the template for PCR amplification of fragments of at least 8 kb. PMID:27240644

  7. GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA.

    PubMed

    Blount, Benjamin A; Driessen, Maureen R M; Ellis, Tom

    2016-01-01

    Existing yeast genomic DNA extraction methods are not ideally suited to extensive screening of colonies by PCR, due to being too lengthy, too laborious or yielding poor quality DNA and inconsistent results. We developed the GC prep method as a solution to this problem. Yeast cells from colonies or liquid cultures are lysed by vortex mixing with glass beads and then boiled in the presence of a metal chelating resin. In around 12 minutes, multiple samples can be processed to extract high yields of genomic DNA. These preparations perform as effectively in PCR screening as DNA purified by organic solvent methods, are stable for up to 1 year at room temperature and can be used as the template for PCR amplification of fragments of at least 8 kb. PMID:27240644

  8. High-resolution DNA-binding specificity analysis of yeast transcription factors

    PubMed Central

    Zhu, Cong; Byers, Kelsey J.R.P.; McCord, Rachel Patton; Shi, Zhenwei; Berger, Michael F.; Newburger, Daniel E.; Saulrieta, Katrina; Smith, Zachary; Shah, Mita V.; Radhakrishnan, Mathangi; Philippakis, Anthony A.; Hu, Yanhui; De Masi, Federico; Pacek, Marcin; Rolfs, Andreas; Murthy, Tal; LaBaer, Joshua; Bulyk, Martha L.

    2009-01-01

    Transcription factors (TFs) regulate the expression of genes through sequence-specific interactions with DNA-binding sites. However, despite recent progress in identifying in vivo TF binding sites by microarray readout of chromatin immunoprecipitation (ChIP-chip), nearly half of all known yeast TFs are of unknown DNA-binding specificities, and many additional predicted TFs remain uncharacterized. To address these gaps in our knowledge of yeast TFs and their cis regulatory sequences, we have determined high-resolution binding profiles for 89 known and predicted yeast TFs, over more than 2.3 million gapped and ungapped 8-bp sequences (“k-mers”). We report 50 new or significantly different direct DNA-binding site motifs for yeast DNA-binding proteins and motifs for eight proteins for which only a consensus sequence was previously known; in total, this corresponds to over a 50% increase in the number of yeast DNA-binding proteins with experimentally determined DNA-binding specificities. Among other novel regulators, we discovered proteins that bind the PAC (Polymerase A and C) motif (GATGAG) and regulate ribosomal RNA (rRNA) transcription and processing, core cellular processes that are constituent to ribosome biogenesis. In contrast to earlier data types, these comprehensive k-mer binding data permit us to consider the regulatory potential of genomic sequence at the individual word level. These k-mer data allowed us to reannotate in vivo TF binding targets as direct or indirect and to examine TFs' potential effects on gene expression in ∼1700 environmental and cellular conditions. These approaches could be adapted to identify TFs and cis regulatory elements in higher eukaryotes. PMID:19158363

  9. Identification of food and beverage spoilage yeasts from DNA sequence analyses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Detection, identification, and classification of yeasts has undergone a major transformation in the last decade and a half following application of gene sequence analyses and genome comparisons. Development of a database (barcode) of easily determined DNA sequences from domains 1 and 2 (D1/D2) of th...

  10. Proficient Replication of the Yeast Genome by a Viral DNA Polymerase.

    PubMed

    Stodola, Joseph L; Stith, Carrie M; Burgers, Peter M

    2016-05-27

    DNA replication in eukaryotic cells requires minimally three B-family DNA polymerases: Pol α, Pol δ, and Pol ϵ. Pol δ replicates and matures Okazaki fragments on the lagging strand of the replication fork. Saccharomyces cerevisiae Pol δ is a three-subunit enzyme (Pol3-Pol31-Pol32). A small C-terminal domain of the catalytic subunit Pol3 carries both iron-sulfur cluster and zinc-binding motifs, which mediate interactions with Pol31, and processive replication with the replication clamp proliferating cell nuclear antigen (PCNA), respectively. We show that the entire N-terminal domain of Pol3, containing polymerase and proofreading activities, could be effectively replaced by those from bacteriophage RB69, and could carry out chromosomal DNA replication in yeast with remarkable high fidelity, provided that adaptive mutations in the replication clamp PCNA were introduced. This result is consistent with the model that all essential interactions for DNA replication in yeast are mediated through the small C-terminal domain of Pol3. The chimeric polymerase carries out processive replication with PCNA in vitro; however, in yeast, it requires an increased involvement of the mutagenic translesion DNA polymerase ζ during DNA replication. PMID:27072134

  11. DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts.

    PubMed Central

    Murakami, H; Nurse, P

    2000-01-01

    The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast. PMID:10861204

  12. DNA replication components as regulators of epigenetic inheritance--lesson from fission yeast centromere.

    PubMed

    He, Haijin; Gonzalez, Marlyn; Zhang, Fan; Li, Fei

    2014-06-01

    Genetic information stored in DNA is accurately copied and transferred to subsequent generations through DNA replication. This process is accomplished through the concerted actions of highly conserved DNA replication components. Epigenetic information stored in the form of histone modifications and DNA methylation, constitutes a second layer of regulatory information important for many cellular processes, such as gene expression regulation, chromatin organization, and genome stability. During DNA replication, epigenetic information must also be faithfully transmitted to subsequent generations. How this monumental task is achieved remains poorly understood. In this review, we will discuss recent advances on the role of DNA replication components in the inheritance of epigenetic marks, with a particular focus on epigenetic regulation in fission yeast. Based on these findings, we propose that specific DNA replication components function as key regulators in the replication of epigenetic information across the genome. PMID:24691906

  13. Analysis of ssDNA Gaps and DSBs in Genetically Unstable Yeast Cultures

    PubMed Central

    Peng, Jie; Raghuraman, M.K.; Feng, Wenyi

    2015-01-01

    DNA replication defects are an underlying cause of genome instability, which could stem from alterations in replication intermediates such as extensive single-stranded DNA (ssDNA). Under replication stress, ssDNA is a precursor of the ultimate double-strand breaks (DSBs). Indeed, mutations that render the cell incapable of mediating and protecting the replication forks produce ssDNA genome-wide at high frequency and cause lethality when encountering DNA damage or replication perturbation. Here we describe two related microarray-based methods to query genetically unstable yeast cultures, such as the mec1 and rad53 mutants. These mutants are defective in central protein kinases in the checkpoint pathway. To induce ssDNA and DSB formation in these mutants, we utilize hydroxyurea, a drug that causes nucleotide shortage in the cell. PMID:24906332

  14. A modified procedure for isolation of yeast mitochondrial DNA.

    PubMed

    Nedeva, Trayana; Petrova, Ventzislava; Hristozova, Tsonka; Kujumdzieva, Anna

    2002-01-01

    A modified, rapid and inexpensive method for preparation of mitochondrial DNA (mtDNA), suitable for molecular analysis is proposed. It comprises batch cultivation of Saccharomyces cerevisiae strain NBIMCC 583 on a simple nutrient medium at 28 degrees C; permeabialization of cells from late exponential growth phase with cetyltrimethylamonnium bromide, mechanical disintegration of the cell wall; preparation of a mitochondrial fraction and subsequent isolation and purification of mtDNA. The amount and the purity of the obtained mtDNA have been checked and its application for molecular analysis proven. The main advantages of the proposed procedure for isolation of mtDNA are introduction of simple nutrient medium, replacement of the enzymatic lysis of the cell wall by the cheaper mechanical one, avoidance of ultracentrifugation steps and use of harmful chemical substances. PMID:12440743

  15. Transcription-dependent DNA transactions in the mitochondrial genome of a yeast hypersuppressive petite mutant.

    PubMed

    Van Dyck, E; Clayton, D A

    1998-05-01

    Mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae contains highly conserved sequences, called rep/ori, that are associated with several aspects of its metabolism. These rep/ori sequences confer the transmission advantage exhibited by a class of deletion mutants called hypersuppressive petite mutants. In addition, because they share features with the mitochondrial leading-strand DNA replication origin of mammals, rep/ori sequences have also been proposed to participate in mtDNA replication initiation. Like the mammalian origins, where transcription is used as a priming mechanism for DNA synthesis, yeast rep/ori sequences contain an active promoter. Although transcription is required for maintenance of wild-type mtDNA in yeast, the role of the rep/ori promoter as a cis-acting element involved in the replication of wild-type mtDNA is unclear, since mitochondrial deletion mutants need neither transcription nor a rep/ori sequence to maintain their genome. Similarly, transcription from the rep/ori promoter does not seem to be necessary for biased inheritance of mtDNA. As a step to elucidate the function of the rep/ori promoter, we have attempted to detect transcription-dependent DNA transactions in the mtDNA of a hypersuppressive petite mutant. We have examined the mtDNA of the well-characterized petite mutant a-1/1R/Z1, whose repeat unit shelters the rep/ori sequence ori1, in strains carrying either wild-type or null alleles of the nuclear genes encoding the mitochondrial transcription apparatus. Complex DNA transactions were detected that take place around GC-cluster C, an evolutionarily conserved GC-rich sequence block immediately downstream from the rep/ori promoter. These transactions are strictly dependent upon mitochondrial transcription. PMID:9566917

  16. The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

    PubMed Central

    Bakkaiova, Jana; Arata, Kosuke; Matsunobu, Miki; Ono, Bungo; Aoki, Tomoyo; Lajdova, Dana; Nebohacova, Martina; Nosek, Jozef; Miyakawa, Isamu

    2014-01-01

    Mitochondrial DNA (mtDNA) is highly compacted into DNA-protein structures termed mitochondrial nucleoids (mt-nucleoids). The key mt-nucleoid components responsible for mtDNA condensation are HMG box-containing proteins such as mammalian mitochondrial transcription factor A (TFAM) and Abf2p of the yeast Saccharomyces cerevisiae. To gain insight into the function and organization of mt-nucleoids in strictly aerobic organisms, we initiated studies of these DNA-protein structures in Yarrowia lipolytica. We identified a principal component of mt-nucleoids in this yeast and termed it YlMhb1p (Y. lipolytica mitochondrial HMG box-containing protein 1). YlMhb1p contains two putative HMG boxes contributing both to DNA binding and to its ability to compact mtDNA in vitro. Phenotypic analysis of a Δmhb1 strain lacking YlMhb1p resulted in three interesting findings. First, although the mutant exhibits clear differences in mt-nucleoids accompanied by a large decrease in the mtDNA copy number and the number of mtDNA-derived transcripts, its respiratory characteristics and growth under most of the conditions tested are indistinguishable from those of the wild-type strain. Second, our results indicate that a potential imbalance between subunits of the respiratory chain encoded separately by nuclear DNA and mtDNA is prevented at a (post)translational level. Third, we found that mtDNA in the Δmhb1 strain is more prone to mutations, indicating that mtHMG box-containing proteins protect the mitochondrial genome against mutagenic events. PMID:24972935

  17. Positional dependence of transcriptional inhibition by DNA torsional stress in yeast chromosomes.

    PubMed

    Joshi, Ricky S; Piña, Benjamin; Roca, Joaquim

    2010-02-17

    How DNA helical tension is constrained along the linear chromosomes of eukaryotic cells is poorly understood. In this study, we induced the accumulation of DNA (+) helical tension in Saccharomyces cerevisiae cells and examined how DNA transcription was affected along yeast chromosomes. The results revealed that, whereas the overwinding of DNA produced a general impairment of transcription initiation, genes situated at <100 kb from the chromosomal ends gradually escaped from the transcription stall. This novel positional effect seemed to be a simple function of the gene distance to the telomere: It occurred evenly in all 32 chromosome extremities and was independent of the atypical structure and transcription activity of subtelomeric chromatin. These results suggest that DNA helical tension dissipates at chromosomal ends and, therefore, provides a functional indication that yeast chromosome extremities are topologically open. The gradual escape from the transcription stall along the chromosomal flanks also indicates that friction restrictions to DNA twist diffusion, rather than tight topological boundaries, might suffice to confine DNA helical tension along eukaryotic chromatin. PMID:20057354

  18. Positional dependence of transcriptional inhibition by DNA torsional stress in yeast chromosomes

    PubMed Central

    Joshi, Ricky S; Piña, Benjamin; Roca, Joaquim

    2010-01-01

    How DNA helical tension is constrained along the linear chromosomes of eukaryotic cells is poorly understood. In this study, we induced the accumulation of DNA (+) helical tension in Saccharomyces cerevisiae cells and examined how DNA transcription was affected along yeast chromosomes. The results revealed that, whereas the overwinding of DNA produced a general impairment of transcription initiation, genes situated at <100 kb from the chromosomal ends gradually escaped from the transcription stall. This novel positional effect seemed to be a simple function of the gene distance to the telomere: It occurred evenly in all 32 chromosome extremities and was independent of the atypical structure and transcription activity of subtelomeric chromatin. These results suggest that DNA helical tension dissipates at chromosomal ends and, therefore, provides a functional indication that yeast chromosome extremities are topologically open. The gradual escape from the transcription stall along the chromosomal flanks also indicates that friction restrictions to DNA twist diffusion, rather than tight topological boundaries, might suffice to confine DNA helical tension along eukaryotic chromatin. PMID:20057354

  19. DNA strand annealing is promoted by the yeast Rad52 protein.

    PubMed Central

    Mortensen, U H; Bendixen, C; Sunjevaric, I; Rothstein, R

    1996-01-01

    The Saccharomyces cerevisiae RAD52 gene plays a pivotal role in genetic recombination. Here we demonstrate that yeast Rad52 is a DNA binding protein. To show that the interaction between Rad52 and DNA is direct and not mediated by other yeast proteins and to facilitate protein purification, a recombinant expression system was developed. The recombinant protein can bind both single- and double-stranded DNA and the addition of either Mg2+ or ATP does not enhance the binding of single-stranded DNA. Furthermore, a DNA binding domain was found in the evolutionary conserved N terminus of the protein. More importantly, we show that the protein stimulates DNA annealing even in the presence of a large excess of nonhomologous DNA. Rad52-promoted annealing follows second-order kinetics and the rate is 3500-fold faster than that of the spontaneous reaction. How this annealing activity relates to the genetic phenotype associated with rad52 mutant cells is discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8855248

  20. Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks.

    PubMed

    Zhou, Chunshui; Elia, Andrew E H; Naylor, Maria L; Dephoure, Noah; Ballif, Bryan A; Goel, Gautam; Xu, Qikai; Ng, Aylwin; Chou, Danny M; Xavier, Ramnik J; Gygi, Steven P; Elledge, Stephen J

    2016-06-28

    The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast. PMID:27298372

  1. Repair of uv damaged DNA: Genes and proteins of yeast and human. Progress report, November 1, 1991--April 15, 1992

    SciTech Connect

    Prakash, L.

    1992-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, and to study the human homologs of yeast excision repair and postreplication repair proteins progress is described.

  2. A Novel Mutation in DNA Topoisomerase I of Yeast Causes DNA Damage and Rad9-Dependent Cell Cycle Arrest

    PubMed Central

    Levin, N. A.; Bjornsti, M. A.; Fink, G. R.

    1993-01-01

    DNA topoisomerases, enzymes that alter the superhelicity of DNA, have been implicated in such critical cellular functions as transcription, DNA replication, and recombination. In the yeast Saccharomyces cerevisiae, a null mutation in the gene encoding topoisomerase I (TOP1) causes elevated levels of mitotic recombination in the ribosomal DNA (rDNA), but has little effect on growth. We have isolated a missense mutation in TOP1 that causes mitotic hyper-recombination not only in the rDNA, but also at other loci, in addition to causing a number of other unexpected phenotypes. This topoisomerase I mutation (top1-103) causes slow growth, constitutive expression of DNA damage-inducible genes, and inviability in the absence of the double-strand break repair system. Overexpression of top1-103 causes RAD9-dependent cell cycle arrest in G(2). We show that the Top1-103 enzyme nicks DNA in vitro, suggesting that it damages DNA directly. We propose that Top1-103 mimics the action of wild-type topoisomerase I in the presence of the anti-tumor drug, camptothecin. PMID:8385050

  3. Satellite DNA from the brine shrimp Artemia affects the expression of a flanking gene in yeast.

    PubMed

    Maiorano, D; Cece, R; Badaracco, G

    1997-04-11

    We have previously revealed that in the brine shrimp Artemia franciscana an AluI DNA family of repeats, 113 bp in length, is the major component of the constitutive heterochromatin and that this repetitive DNA shows a stable curvature that confers a solenoidal geometry on the double helix in vitro. It was suggested that this particular structure may play a relevant role in determining the condensation of the heterochromatin. In this report we have cloned hexamers of highly-repetitive sequence (AluI-satellite DNA) in proximity to a yeast lacZ reporter gene on a plasmid. We find that the expression of the reporter gene is affected by the presence of this DNA in a dose- and orientation-dependent manner in the yeast, S. cerevisiae. We show that this effect is not dependent on under-replication or re-arrangements of the repetitive DNA in the cell but is due to decreased expression of the reporter gene. Our results indicate that the AluI-satellite DNA of Artemia per se is able to influence gene expression. PMID:9161405

  4. Crystal Structure of Yeast DNA Polymerase ε Catalytic Domain

    PubMed Central

    Jain, Rinku; Rajashankar, Kanagalaghatta R.; Buku, Angeliki; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2014-01-01

    DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Here we report the ternary structure of the Polε catalytic subunit (Pol2) bound to a nascent G:C base pair (Pol2G:C). Pol2G:C has a typical B-family polymerase fold and embraces the template-primer duplex with the palm, fingers, thumb and exonuclease domains. The overall arrangement of domains is similar to the structure of Pol2T:A reported recently, but there are notable differences in their polymerase and exonuclease active sites. In particular, we observe Ca2+ ions at both positions A and B in the polymerase active site and also observe a Ca2+ at position B of the exonuclease site. We find that the contacts to the nascent G:C base pair in the Pol2G:C structure are maintained in the Pol2T:A structure and reflect the comparable fidelity of Pol2 for nascent purine-pyrimidine and pyrimidine-purine base pairs. We note that unlike that of Pol3, the shape of the nascent base pair binding pocket in Pol2 is modulated from the major grove side by the presence of Tyr431. Together with Pol2T:A, our results provide a framework for understanding the structural basis of high fidelity DNA synthesis by Pol2. PMID:24733111

  5. Fission Yeast Pxd1 Promotes Proper DNA Repair by Activating Rad16XPF and Inhibiting Dna2

    PubMed Central

    Zhang, Jia-Min; Liu, Xiao-Man; Ding, Yue-He; Xiong, Liang-Yao; Ren, Jing-Yi; Zhou, Zhi-Xiong; Wang, Hai-Tao; Zhang, Mei-Jun; Yu, Yang; Dong, Meng-Qiu; Du, Li-Lin

    2014-01-01

    Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16XPF-Swi10ERCC1 and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 3′ endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 5′ endonuclease activity of Dna2. Pxd1 is required for Rad16-Swi10 to function in single-strand annealing, mating-type switching, and the removal of Top1-DNA adducts. Meanwhile, Pxd1 attenuates DNA end resection mediated by the Rqh1-Dna2 pathway. Disabling the Dna2-inhibitory activity of Pxd1 results in enhanced use of a break-distal repeat sequence in single-strand annealing and a greater loss of genetic information. We propose that Pxd1 promotes proper DNA repair by differentially regulating two structure-specific nucleases. PMID:25203555

  6. Yeast telomeres exert a position effect on recombination between internal tracts of yeast telomeric DNA

    PubMed Central

    Stavenhagen, Jeffrey B.; Zakian, Virginia A.

    1998-01-01

    In Saccharomyces cerevisiae, proximity to a telomere affects both transcription and replication of adjacent DNA. In this study, we show that telomeres also impose a position effect on mitotic recombination. The rate of recombination between directly repeated tracts of telomeric C1–3A/TG1–3 DNA was reduced severely by proximity to a telomere. In contrast, recombination of two control substrates was not affected by telomere proximity. Thus, unlike position effects on transcription or replication, inhibition of recombination was sequence specific. Moreover, the repression of recombination was not under the same control as transcriptional repression (telomere position effect; TPE), as mutations in genes essential for TPE did not alleviate telomeric repression of recombination. The reduction in recombination between C1–3A/TG1–3 tracts near the telomere was caused by an absence of Rad52p-dependent events as well as a reduction in Rad1p-dependent events. The sequence-specific repression of recombination near the telomere was eliminated in cells that overexpressed the telomere-binding protein Rap1p, a condition that also increased recombination between C1–3A/TG1–3 tracts at internal positions on the chromosome. We propose that the specific inhibition between C1–3A/TG1–3 tracts near the telomere occurs through the action of a telomere-specific end-binding protein that binds to the single-strand TG1–3 tail generated during the processing of recombination intermediates. The recombination inhibitor protein may also block recombination between endogenous telomeres. PMID:9765206

  7. Replication dynamics in fission and budding yeasts through DNA polymerase tracking.

    PubMed

    Vázquez, Enrique; Antequera, Francisco

    2015-10-01

    The dynamics of eukaryotic DNA polymerases has been difficult to establish because of the difficulty of tracking them along the chromosomes during DNA replication. Recent work has addressed this problem in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae through the engineering of replicative polymerases to render them prone to incorporating ribonucleotides at high rates. Their use as tracers of the passage of each polymerase has provided a picture of unprecedented resolution of the organization of replicons and replication origins in the two yeasts and has uncovered important differences between them. Additional studies have found an overlapping distribution of DNA polymorphisms and the junctions of Okazaki fragments along mononucleosomal DNA. This sequence instability is caused by the premature release of polymerase δ and the retention of non proof-read DNA tracts replicated by polymerase α. The possible implementation of these new experimental approaches in multicellular organisms opens the door to the analysis of replication dynamics under a broad range of genetic backgrounds and physiological or pathological conditions. PMID:26293347

  8. Astragalin from Cassia alata induces DNA adducts in vitro and repairable DNA damage in the yeast Saccharomyces cerevisiae.

    PubMed

    Saito, Samuel; Silva, Givaldo; Santos, Regineide Xavier; Gosmann, Grace; Pungartnik, Cristina; Brendel, Martin

    2012-01-01

    Reverse phase-solid phase extraction from Cassia alata leaves (CaRP) was used to obtain a refined extract. Higher than wild-type sensitivity to CaRP was exhibited by 16 haploid Saccharomyces cerevisiae mutants with defects in DNA repair and membrane transport. CaRP had a strong DPPH free radical scavenging activity with an IC(50) value of 2.27 μg mL(-1) and showed no pro-oxidant activity in yeast. CaRP compounds were separated by HPLC and the three major components were shown to bind to DNA in vitro. The major HPLC peak was identified as kampferol-3-O-β-d-glucoside (astragalin), which showed high affinity to DNA as seen by HPLC-UV measurement after using centrifugal ultrafiltration of astragalin-DNA mixtures. Astragalin-DNA interaction was further studied by spectroscopic methods and its interaction with DNA was evaluated using solid-state FTIR. These and computational (in silico) docking studies revealed that astragalin-DNA binding occurs through interaction with G-C base pairs, possibly by intercalation stabilized by H-bond formation. PMID:22489129

  9. Identification of food and beverage spoilage yeasts from DNA sequence analyses.

    PubMed

    Kurtzman, Cletus P

    2015-11-20

    Detection, identification and classification of yeasts have undergone major changes in the last decade and a half following application of gene sequence analyses and genome comparisons. Development of a database (barcode) of easily determined DNA sequences from domains 1 and 2 (D1/D2) of the nuclear large subunit rRNA gene and from ITS now permits many laboratories to identify species quickly and accurately, thus replacing the laborious and often inaccurate phenotypic tests previously used. Phylogenetic analysis of gene sequences has resulted in a major revision of yeast systematics resulting in redefinition of nearly all genera. This new understanding of species relationships has prompted a change of rules for naming and classifying yeasts and other fungi, and these new rules are presented in the recently implemented International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). The use of molecular methods for species identification and the impact of Code changes on classification will be discussed, especially in the context of food and beverage spoilage yeasts. PMID:26051959

  10. Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip.

    PubMed

    Chan, Yujia A; Aristizabal, Maria J; Lu, Phoebe Y T; Luo, Zongli; Hamza, Akil; Kobor, Michael S; Stirling, Peter C; Hieter, Philip

    2014-04-01

    DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013. PMID:24743342

  11. Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping

    SciTech Connect

    Weier, Heinz-Ulrich G.; Greulich-Bode, Karin M.; Wu, Jenny; Duell, Thomas

    2009-09-18

    Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.

  12. Genome-Wide Profiling of Yeast DNA:RNA Hybrid Prone Sites with DRIP-Chip

    PubMed Central

    Lu, Phoebe Y. T.; Luo, Zongli; Hamza, Akil; Kobor, Michael S.; Stirling, Peter C.; Hieter, Philip

    2014-01-01

    DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013. PMID:24743342

  13. Surface Etching and DNA Damage Induced by Low-Energy Ion Irradiation in Yeast

    NASA Astrophysics Data System (ADS)

    Liu, Xuelan; Xu, An; Dai, Yin; Yuan, Hang; Yu, Zengliang

    2011-06-01

    Bio-effects of survival and etching damage on cell surface and DNA strand breaks were investigated in the yeast saccharomyces cerevisiae after exposure by nitrogen ion with an energy below 40 keV. The result showed that 16% of trehalose provided definite protection for cells against vacuum stress compared with glycerol. In contrast to vacuum control, significant morphological damage and DNA strand breaks were observed, in yeast cells bombarded with low-energy nitrogen, by scanning electron microscopy (SEM) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) immunofluorescence assays. Moreover, PI (propidium iodide) fluorescent staining indicated that cell integrity could be destroyed by ion irradiation. Cell damage eventually affected cell viability and free radicals were involved in cell damage as shown by DMSO (dimethyl sulfoxide) rescue experiment. Our primary experiments demonstrated that yeast cells can be used as an optional experimental model to study the biological effects of low energy ions and be applied to further investigate the mechanism(s) underlying the bio-effects of eukaryotic cells.

  14. Yeast RAD14 and human xeroderma pigmentosum group A DNA-repair genes encode homologous proteins.

    PubMed

    Bankmann, M; Prakash, L; Prakash, S

    1992-02-01

    Xeroderma pigmentosum (XP), a human autosomal recessive disorder, is characterized by extreme sensitivity to sunlight and high incidence of skin cancers. XP cells are defective in the incision step of excision repair of DNA damaged by ultraviolet light. Cell fusion studies have defined seven XP complementation groups, XP-A to XP-G. Similar genetic complexity of excision repair is observed in the yeast Saccharomyces cerevisiae. Mutations in any one of five yeast genes, RAD1, RAD2, RAD3, RAD4, and RAD10, cause a total defect in incision and an extreme sensitivity to ultraviolet light. Here we report the characterization of the yeast RAD14 gene. The available rad14 point mutant is only moderately ultraviolet-sensitive, and it performs a substantial amount of incision of damaged DNA. Our studies with the rad14 deletion (delta) mutation indicate an absolute requirement of RAD14 in incision. RAD14 encodes a highly hydrophilic protein of 247 amino acids containing zinc-finger motifs, and it is similar to the protein encoded by the human XPAC gene that complements XP group A cell lines. PMID:1741034

  15. Induction and isolation of DNA transformation mutants in the yeast Saccharomyces cerevisiae

    SciTech Connect

    Hegerich, P.A.; Bruschi, C.V.

    1987-01-01

    The objective of this research was to induce and isolate mutants of the yeast Saccharomyces cerevisiae which have become transformable by purified plasmid DNA. Non-transformable yeast cells were mutagenized by ultraviolet light using a 65% lethal dose (480 ergs/mm/sup 2/). After a period of overnight liquid holding recovery, the irradiated cells were subjected to DNA transformation using our CaCl/sub 2/ protocol with the multi-marker shuttle plasmid pBB carrying the LEU 2 leucine gene. Following transformation the colonies that grew on selective leucineless medium were identified and subjected to further genetic analysis. From a total of 1 x 10/sup 9/ cells the authors have isolated 7 colonies deriving from putative mutants that have acquired the capability to uptake plasmid DNA. The transformants were cured from the plasmid by its mitotic loss on non-selective medium, then re-transformed to verify their genetic competence to give rise to a number of transformants comparable to transformable strains. We have identified and isolated one mutant, coded trs-1, which is able to reproduce a frequency of transformation comparable with the tranformable control. They, therefore, conclude that this mutant is specific for plasmid DNA transformation and that the mutation is mitotically stable.

  16. Genetic transformation of the yeast Dekkera/Brettanomyces bruxellensis with non-homologous DNA.

    PubMed

    Miklenić, Marina; Štafa, Anamarija; Bajić, Ana; Žunar, Bojan; Lisnić, Berislav; Svetec, Ivan-Krešimir

    2013-05-01

    Yeast Dekkera/Brettanomyces bruxellensis is probably the most common contaminant in wineries and ethanol production processes. The considerable economic losses caused by this yeast, but also its ability to produce and tolerate high ethanol concentrations, make it an attractive subject for research with potential for industrial applications. Unfortunately, efforts to understand the biology of D. bruxellensis and facilitate its broader use in industry are hampered by the lack of adequate procedures for delivery of exogenous DNA into this organism. Here we describe the development of transformation protocols (spheroplast transformation, LiAc/PEG method, and electroporation) and report the first genetic transformation of yeast D. bruxellensis. A linear heterologous DNA fragment carrying the kanMX4 sequence was used for transformation, which allowed transformants to be selected on plates containing geneticin. We found the spheroplast transformation method using 1M sorbitol as osmotic stabilizer to be inappropriate because sorbitol strikingly decreases the plating efficiency of both D. bruxellensis spheroplast and intact cells. However, we managed to modify the LiAc/ PEG transformation method and electroporation to accommodate D. bruxellensis transformation, achieving efficiencies of 0.6-16 and 10-20 transformants/microg DNA, respectively. The stability of the transformants ranged from 93.6% to 100%. All putative transformants were analyzed by Southern blot using the kanMX4 sequence as a hybridization probe, which confirmed that the transforming DNA fragment had integrated into the genome. The results of the molecular analysis were consistent with the expected illegitimate integration of a heterologous transforming fragment. PMID:23648858

  17. In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse.

    PubMed

    Morselli, Marco; Pastor, William A; Montanini, Barbara; Nee, Kevin; Ferrari, Roberto; Fu, Kai; Bonora, Giancarlo; Rubbi, Liudmilla; Clark, Amander T; Ottonello, Simone; Jacobsen, Steven E; Pellegrini, Matteo

    2015-01-01

    Methylation of cytosines (5(me)C) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5(me)C levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo. PMID:25848745

  18. Connections between RNA splicing and DNA intron mobility in yeast mitochondria: RNA maturase and DNA endonuclease switching experiments.

    PubMed Central

    Goguel, V; Delahodde, A; Jacq, C

    1992-01-01

    The intron-encoded proteins bI4 RNA maturase and aI4 DNA endonuclease can be faithfully expressed in yeast cytoplasm from engineered forms of their mitochondrial coding sequences. In this work we studied the relationships between these two activities associated with two homologous intron-encoded proteins: the bI4 RNA maturase encoded in the fourth intron of the cytochrome b gene and the aI4 DNA endonuclease (I-SceII) encoded in the fourth intron of the gene coding for the subunit I of cytochrome oxidase. Taking advantage of both the high recombinogenic properties of yeast and the similarities between the two genes, we constructed in vivo a family of hybrid genes carrying parts of both RNA maturase and DNA endonuclease coding sequences. The presence of a sequence coding for a mitochondrial targeting peptide upstream from these hybrid genes allowed us to study the properties of their translation products within the mitochondria in vivo. We thus could analyze the ability of the recombinant proteins to complement RNA maturase deficiencies in different strains. Many combinations of the two parental intronic sequences were found in the recombinants. Their structural and functional analysis revealed the following features. (i) The N-terminal half of the bI4 RNA maturase could be replaced in total by its equivalent from the aI4 DNA endonuclease without affecting the RNA maturase activity. In contrast, replacing the C-terminal half of the bI4 RNA maturase with its equivalent from the aI4 DNA endonuclease led to a very weak RNA maturase activity, indicating that this region is more differentiated and linked to the maturase activity. (ii) None of the hybrid proteins carrying an RNA maturase activity kept the DNA endonuclease activity, suggesting that the latter requires the integrity of the aI4 protein. These observations are interesting because the aI4 DNA endonuclease is known to promote the propagation, at the DNA level, of the aI4 intron, whereas the bI4 RNA maturase

  19. Yeast DNA-repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA

    SciTech Connect

    Guzder, S.N.; Sung, P.; Prakash, S. ); Prakash, L. )

    1993-06-15

    Xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers due to a defect in excision repair of UV light-damaged DNA. Of the seven XP complementation groups, A--G, group A represents a severe and frequent form of the disease. The Saccharomyces cerevisiae RAD14 gene is a homolog of the XP-A correcting (XPAC) gene. Like XP-A cells, rad14-null mutants are defective in the incision step of excision repair of UV-damaged DNA. The authors have purified RAD14 protein to homogeneity from extract of a yeast strain genetically tailored to overexpress RAD14. As determined by atomic emission spectroscopy, RAD14 contains one zinc atom. They also show in vitro that RAD14 binds zinc but does not bind other divalent metal ions. In DNA mobility-shift assays, RAD14 binds specifically to UV-damaged DNA. Removal of cyclobutane pyrimidine dimers from damaged DNA by enzymatic photoreactivation has no effect on binding, strongly suggesting that RAD14 recognizes pyrimidine(6-4)pyrimidone photoproduct sites. These findings indicate that RAD14 functions in damage recognition during excision repair. 37 refs., 4 figs.

  20. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis.

    PubMed Central

    Carle, G F; Olson, M V

    1984-01-01

    A simple agarose-gel apparatus has been developed that allows the separation of DNA molecules in the size range from 50 kb to well over 750 kb, the largest size for which size standards were available. The apparatus is based on the recent discovery that large DNA molecules are readily fractionated on agarose gels if they are alternately subjected to two approximately orthogonal electric fields. The switching time, which was on the order of 20-50 sec in our experiments, can be adjusted to optimize fractionation in a given size range. The resolution of the technique is sufficient to allow the fractionation of a sample of self-ligated lambda DNA into a ladder of approximately 15 bands, spaced at 50 kb intervals. We have applied the technique to the fractionation of yeast DNA into 11 distinct bands, several of which have been shown by DNA-DNA hybridization to hybridize uniquely to different chromosome-specific hybridization probes. In this paper, we describe the design of the apparatus, the electrophoretic protocol, and the sample-handling procedures that we have employed. Images PMID:6379602

  1. Two polypeptide chains in yeast transcription factor tau interact with DNA

    SciTech Connect

    Gabrielsen, O.S.; Marzouki, N.; Ruet, A.; Sentenac, A.; Fromageot, P.

    1989-05-05

    Yeast transcription factor tau interacts with the A and B blocks of the intragenic promoter of tRNA genes. The structure of tau was investigated by identifying the polypeptide chains specifically complexed to the tRNA3Glu gene. Highly purified factor, obtained by an improved purification procedure, contained several polypeptide chains, four of which (Mr = 145,000, 135,000, 100,000 and 65,000) comigrated with tau-DNA complex by polyacrylamide gel electrophoresis. Antibodies raised against the 145- and 100-kDa components altered the migration of tau-DNA complexes in band shift assays and inhibited tRNA synthesis in a reconstituted transcription system. These components are immunologically unrelated proteins. By UV cross-linking to /sup 32/P-body-labeled tDNA followed by extensive DNase treatment, two polypeptides of the same size (145 and 100 kDa) were found to be radioactively labeled. Factor tau, therefore, appears to be a multisubunit DNA-binding protein with two distinct polypeptides contributing to DNA recognition. Limited proteolysis of tau generated a protease-resistant tau B (tau B) domain that binds solely to the B block. tau B-tDNA complexes were recognized by anti-145 IgG and contained a 120-kDa polypeptide that could originate from the 145-kDa component by proteolysis. These results strongly suggest that the 145-kDa polypeptide belongs to tau B and is responsible for B block binding.

  2. Effect of storage and processing on plasmid, yeast and plant genomic DNA stability in juice from genetically modified oranges.

    PubMed

    Weiss, Julia; Ros-Chumillas, Maria; Peña, Leandro; Egea-Cortines, Marcos

    2007-01-30

    Recombinant DNA technology is an important tool in the development of plant varieties with new favourable features. There is strong opposition towards this technology due to the potential risk of horizontal gene transfer between genetically modified plant material and food-associated bacteria, especially if genes for antibiotic resistance are involved. Since horizontal transfer efficiency depends on size and length of homologous sequences, we investigated the effect of conditions required for orange juice processing on the stability of DNA from three different origins: plasmid DNA, yeast genomic DNA and endogenous genomic DNA from transgenic sweet orange (C. sinensis L. Osb.). Acidic orange juice matrix had a strong degrading effect on plasmid DNA which becomes apparent in a conformation change from supercoiled structure to nicked, linear structure within 5h of storage at 4 degrees C. Genomic yeast DNA was degraded during exposure to acidic orange juice matrix within 4 days, and also the genomic DNA of C. sinensis suffered degradation within 2 days of storage as indicated by amplification results from transgene markers. Standard pasteurization procedures affected DNA integrity depending on the method and time used. Our data show that the current standard industrial procedures to pasteurize orange juice as well as its acidic nature causes a strong degradation of both yeast and endogenous genomic DNA below sizes reported to be suitable for horizontal gene transfer. PMID:17064805

  3. A direct droplet digital PCR method for quantification of residual DNA in protein drugs produced in yeast cells.

    PubMed

    Hussain, Musaddeq; Fantuzzo, Rebecca; Mercorelli, Suzanne; Cullen, Constance

    2016-05-10

    Yeast cells, in particular Pichia pastoris, are the host cell of choice for manufacturing several protein therapeutic agents in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and the residual DNA after the purification process of the drug must be monitored to ensure drug purity and safety. Currently, real-time PCR (qPCR) based methods are widely employed for quantification of host residual DNA. At the same time the digital PCR technology is coming into prominence with promise of higher sensitivity. Here we report a method where the protein drug is directly added to the droplet digital PCR (ddPCR) reaction including yeast-specific primers and fluorescent-tagged probe and nanoliter-sized droplets are generated. The droplets are then subjected to PCR followed by analysis for fluorescence. This Pichia residual DNA direct ddPCR method for yeast can be used to test higher amount of drug compared to the corresponding qPCR method thereby increasing sensitivity, retaining high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with three batches of a recombinant human IgG1-Fc-based drug (RP-1) and with commercially available human insulin, both manufactured in yeast cells. This method simplifies the residual DNA quantification protocol by eliminating DNA extraction or protease digestion and eliminates use of DNA standards in day-to-day running of the method. PMID:26896631

  4. The DNA damage checkpoint allows recombination between divergent DNA sequences in budding yeast

    PubMed Central

    George, Carolyn M.; Lyndaker, Amy M.; Alani, Eric

    2011-01-01

    In the early steps of homologous recombination, single-stranded DNA (ssDNA) from a broken chromosome invades homologous sequence located in a sister or homolog donor. In genomes that contain numerous repetitive DNA elements or gene paralogs, recombination can potentially occur between non-allelic/divergent (homeologous) sequences that share sequence identity. Such recombination events can lead to lethal chromosomal deletions or rearrangements. However, homeologous recombination events can be suppressed through rejection mechanisms that involve recognition of DNA mismatches in heteroduplex DNA by mismatch repair factors, followed by active unwinding of the heteroduplex DNA by helicases. Because factors required for heteroduplex rejection are hypothesized to be targets and/or effectors of the DNA damage response (DDR), a cell cycle control mechanism that ensures timely and efficient repair, we tested whether the DDR, and more specifically, the RAD9 gene, had a role in regulating rejection. We performed these studies using a DNA repair assay that measures repair by single-strand annealing (SSA) of a double-strand break (DSB) using homeologous DNA templates. We found that repair of homeologous DNA sequences, but not identical sequences, induced a RAD9- dependent cell cycle delay in the G2 stage of the cell cycle. Repair through a divergent DNA template occurred more frequently in RAD9 compared to rad9Δ strains. However, repair in rad9Δ mutants could be restored to wild-type levels if a G2 delay was induced by nocodazole. These results suggest that cell cycle arrest induced by the Rad9-dependent DDR allows repair between divergent DNA sequences despite the potential for creating deleterious genome rearrangements, and illustrates the importance of additional cellular mechanisms that act to suppress recombination between divergent DNA sequences. PMID:21978436

  5. Stable yeast transformants that secrete functional. cap alpha. -amylase encoded by cloned mouse pancreatic cDNA

    SciTech Connect

    Filho, S.A.; Galembeck, E.V.; Faria, J.B.; Frascino, A.C.S.

    1986-04-01

    Mouse pancreatic ..cap alpha..-amylase complementary DNA was inserted into a yeast shuttle vector after the Saccharomyces cerevisiae MF..cap alpha..1 promoter and secretion signals coding sequences. When transformed with the recombinant plasmid, S. cerevisiae cells were able to synthesize and secrete functional ..cap alpha..-amylase, efficiently hydrolyzing starch present in the culture medium. Stable amylolytic cells were obtained from different yeast strains. This work represents a significant step towards producing yeast that can convert starchy materials directly to ethanol.

  6. Yeast Rad17/Mec3/Ddc1: A sliding clamp for the DNA damage checkpoint

    PubMed Central

    Majka, Jerzy; Burgers, Peter M. J.

    2003-01-01

    The Saccharomyces cerevisiae Rad24 and Rad17 checkpoint proteins are part of an early response to DNA damage in a signal transduction pathway leading to cell cycle arrest. Rad24 interacts with the four small subunits of replication factor C (RFC) to form the RFC-Rad24 complex. Rad17 forms a complex with Mec3 and Ddc1 (Rad17/3/1) and shows structural similarities with the replication clamp PCNA. This parallelism with a clamp-clamp loader system that functions in DNA replication has led to the hypothesis that a similar clamp-clamp loader relationship exists for the DNA damage response system. We have purified the putative checkpoint clamp loader RFC-Rad24 and the putative clamp Rad17/3/1 from a yeast overexpression system. Here, we provide experimental evidence that, indeed, the RFC-Rad24 clamp loader loads the Rad17/3/1 clamp around partial duplex DNA in an ATP-dependent process. Furthermore, upon ATP hydrolysis, the Rad17/3/1 clamp is released from the clamp loader and can slide across more than 1 kb of duplex DNA, a process which may be well suited for a search for damage. Rad17/3/1 showed no detectable exonuclease activity. PMID:12604797

  7. DNA topoisomerase-targeting antitumor drugs can be studied in yeast.

    PubMed Central

    Nitiss, J; Wang, J C

    1988-01-01

    The antitumor drugs camptothecin and an anilinoacridine, 4'-(9-acridinylamino)-methanesulfon-m-anisidide (mAMSA), which act on DNA topoisomerase I and II, respectively, are shown to inhibit the growth of Saccharomyces cerevisiae mutants selected for their permeability to other inhibitors. In addition to growth inhibition, these drugs induce high levels of homologous recombination and induce the expression of a DNA damage-inducible gene DIN3. Cytotoxicity of the drugs is more pronounced in strains that also carry a rad52 mutation. An analog of mAMSA), which is ineffective as an inhibitor of DNA topoisomerase II in mammalian cells, is also ineffective in eliciting physiological responses in these yeast strains. The physiological effects of camptothecin, but not those of mAMSA, disappear if the TOP1 gene encoding DNA topoisomerase I is disrupted. This shows that DNA topoisomerase I is the sole target of camptothecin cytotoxicity and illustrates that a nonessential enzyme can nevertheless be the target for a cytotoxic drug. PMID:2845409

  8. The role of recombination and RAD52 in mutation of chromosomal DNA transformed into yeast.

    PubMed Central

    Larionov, V; Graves, J; Kouprina, N; Resnick, M A

    1994-01-01

    While transformation is a prominent tool for genetic analysis and genome manipulation in many organisms, transforming DNA has often been found to be unstable relative to established molecules. We determined the potential for transformation-associated mutations in a 360 kb yeast chromosome III composed primarily of unique DNA. Wild-type and rad52 Saccharomyces cerevisiae strains were transformed with either a homologous chromosome III or a diverged chromosome III from S. carlsbergensis. The host strain chromosome III had a conditional centromere allowing it to be lost on galactose medium so that recessive mutations in the transformed chromosome could be identified. Following transformation of a RAD+ strain with the homologous chromosome, there were frequent changes in the incoming chromosome, including large deletions and mutations that do not lead to detectable changes in chromosome size. Based on results with the diverged chromosome, interchromosomal recombinational interactions were the source of many of the changes. Even though rad52 exhibits elevated mitotic mutation rates, the percentage of transformed diverged chromosomes incapable of substituting for the resident chromosome was not increased in rad52 compared to the wild-type strain, indicating that the mutator phenotype does not extend to transforming chromosomal DNA. Based on these results and our previous observation that the incidence of large mutations is reduced during the cloning of mammalian DNA into a rad52 as compared to a RAD+ strain, a rad52 host is well-suited for cloning DNA segments in which gene function must be maintained. Images PMID:7937151

  9. Yeast RNA polymerase II transcription of circular DNA at different degrees of supercoiling.

    PubMed Central

    Pedone, F; Filetici, P; Ballario, P

    1982-01-01

    Purified yeast RNA polymerase II was tested for transcriptional activity as a function of the degree of circular DNA supercoiling. Chimaeric plasmids P30 and P31 both containing inserts from the yeast transposable element TY1 cloned in pBR322 and the vector pBR322 were used as templates. For pBR322 the transcriptional activity increases about 4 fold from the fully relaxed covalently closed circles to the native supercoiled forms, further supercoiling having no effect on transcription. P30 shows a 5 fold increase of transcriptional activity reaching a plateau at the native supercoiled conformation. However, at an intermediate degree of supercoiling (sigma = 0.024), transcription decreases to a value close to zero. P31 too exhibits a conformation (sigma = 0.014) in which there is a drop of transcriptional activity. Furthermore, a 10 fold increase of transcription is obtained at the higher values of superhelix density. Both kinetic and autoradiographic experiments confirm the existence of DNA conformations that can inhibit "in vitro" transcription. Images PMID:6292834

  10. Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation.

    PubMed Central

    Neumann, E; Kakorin, S; Tsoneva, I; Nikolova, B; Tomov, T

    1996-01-01

    Detailed kinetic data suggest that the direct transfer of plasmid DNA (YEp 351, 5.6 kbp, supercoiled, Mr approximately 3.5 x 10(6)) by membrane electroporation of yeast cells (Saccharomyces cerevisiae, strain AH 215) is mainly due to electrodiffusive processes. The rate-limiting step for the cell transformation, however, is a bimolecular DNA-binding interaction in the cell interior. Both the adsorption of DNA, directly measured with [32P]dCTP DNA, and the number of transformants are collinearly enhanced with increasing total concentrations [Dt] and [Cat] of DNA and of calcium, respectively. At [Cat] = 1 mM, the half-saturation or equilibrium constant is KD = 15 +/- 1 nM at 293 K (20 degrees C). The optimal transformation frequency is TFopt = 4.1 +/- 0.4 X 10(-5) if a single exponential pulse of initial field strength E0 = 4 kV cm-1 and decay time constant tauE = 45 ms is applied at [Dt] = 2.7 nM and 10(8) cells in 0.1 ml. The dependence of TF on [Cat] yields the equilibrium constants KCazero = 1.8 +/- 0.2 mM (in the absence of DNA) and K'Ca (at 2.7 nM DNA), comparable with and derived from electrophoresis data. In yeast cells, too, the appearance of a DNA molecule in its whole length in the cell interior is clearly an after-field event. At Eo = 4.0 kV cm-1 and T = 293 K, the flow coefficient of DNA through the porous membrane patches is Kto = 7.0 +/- 0.7 x 10(3)S-1 and the electrodiffusion of DNA is approximately 10 times more effective than simple diffusion: D/D0 approximately 10.3. The mean radius of these pores is rp = 0.39 +/- 0.05 nm, and the mean number of pores per cell (of size ø approximately 5.5 microns) is Np = 2.2 +/- 0.2 x 10(4). The maximal membrane area that is involved in the electrodiffusive penetration of adsorbed DNA into the outer surface of the electroporated cell membrane patches is only 0.023% of the total cell surface. The surface penetration is followed either by additional electrodiffusive or by passive (after-field) diffusive

  11. Characterization of a DNA uptake reaction through the nuclear membrane of isolated yeast nuclei. [Saccharomyces cerevisiae

    SciTech Connect

    Tsuchiya, E.; Shakuto, S.; Miyakawa, T.; Fukui, S.

    1988-02-01

    Isolated yeast nuclei were able to incorporate /sup 3/H-labeled pJDB219 DNA in vitro in the presence of ATP and Mg/sup 2 +/. The number of plasmid molecules incorporated into each nucleus was calculated to be 60 under the conditions we used. Enzyme-histochemical staining of the incorporated biotinylated pJDB219 with streptavidin-biotinylated-peroxidase complex indicated a uniform distribution of the incorporated plasmids within each nucleus. After intranuclear incorporation, substrate pJDB219 DNAs (open and closed circular forms) were changed to the linear form and were weakly digested over the longer incubation period (over 60 min). Facile release of the once-incorporated plasmid DNA was never observable; discharge of the incorporated (/sup 3/H)pJDB219 during a 60-min incubation was less than 5%. The addition of adenylyl-imidodiphosphate, N,N'-dicyclohexylcarbodiimide (DCCD), or quercetin inhibited in vitro DNA uptake reaction. DCCD and quercetin inhibited the nuclear ATPase and apparent protein kinase, respectively; hence, the involvement of these enzymes in the nuclear DNA transport system was suggested.

  12. The fission yeast CENP-B protein Abp1 prevents pervasive transcription of repetitive DNA elements.

    PubMed

    Daulny, Anne; Mejía-Ramírez, Eva; Reina, Oscar; Rosado-Lugo, Jesus; Aguilar-Arnal, Lorena; Auer, Herbert; Zaratiegui, Mikel; Azorin, Fernando

    2016-10-01

    It is well established that eukaryotic genomes are pervasively transcribed producing cryptic unstable transcripts (CUTs). However, the mechanisms regulating pervasive transcription are not well understood. Here, we report that the fission yeast CENP-B homolog Abp1 plays an important role in preventing pervasive transcription. We show that loss of abp1 results in the accumulation of CUTs, which are targeted for degradation by the exosome pathway. These CUTs originate from different types of genomic features, but the highest increase corresponds to Tf2 retrotransposons and rDNA repeats, where they map along the entire elements. In the absence of abp1, increased RNAPII-Ser5P occupancy is observed throughout the Tf2 coding region and, unexpectedly, RNAPII-Ser5P is enriched at rDNA repeats. Loss of abp1 also results in Tf2 derepression and increased nucleolus size. Altogether these results suggest that Abp1 prevents pervasive RNAPII transcription of repetitive DNA elements (i.e., Tf2 and rDNA repeats) from internal cryptic sites. PMID:27345571

  13. Yeast phenotype classifies mammalian protein kinase C cDNA mutants.

    PubMed Central

    Riedel, H; Su, L; Hansen, H

    1993-01-01

    The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of eukaryotic cellular signals. Molecular dissection of their mechanisms of action has been limited in part by the lack of random mutagenesis approaches and by the complexity of signaling pathways in mammalian cells which involve multiple PKC isoforms. Here we present a rapid screen which permits the quantification of mammalian PKC activity phenotypically in the yeast Saccharomyces cerevisiae. Bovine PKC alpha cDNA is functionally expressed in S. cerevisiae. This results in a phorbol ester response: a fourfold increase in the cell doubling time and a substantial decrease in yeast colony size on agar plates. We have expressed pools of bovine PKC alpha cDNAs mutagenized by Bal 31 deletion of internal, amino-terminal, or carboxyl-terminal sequences and have identified three classes of mutants on the basis of their distinct yeast phenotypes. Representatives of each class were analyzed. An internal deletion of amino acids (aa) 172 to 225 displayed ligand-dependent but reduced catalytic activity, an amino-terminal truncation of aa 1 to 153 displayed elevated and ligand-independent activity, and a carboxyl-terminal 26-aa truncation (aa 647 to 672) lacked activity under any conditions. Additional mutations confirmed the distinct functional characteristics of these classes. Our data show that deletion of the V1 and C1 regions results in elevated basal catalytic activity which is still Ca2+ responsive. Internal deletions in the V2 and C2 regions do not abolish phorbol ester or Ca2+ regulation of PKC activity, suggesting that most of the C2 domain is not essential for phorbol ester stimulation and most of the regulatory domain is dispensable for Ca2+ regulation of PKC activity. These distinct activities od the PKC mutants correlate with a specific and proportional yeast phenotype and are quantified on agar plates by yeast colony size. This provides a phenotypic screen which is suitable

  14. Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast.

    PubMed Central

    Polo, S; Ketner, G; Levis, R; Falgout, B

    1997-01-01

    The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The full-length dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E. coli shuttle vector. Full-length cDNA clones were propagated once in E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK2 cells produced typical dengue infection, as judged by cytopathic effects and indirect immunofluorescence. Infectivity was sensitive to RNase digestion and was dependent on the presence of cap analog in the transcription reaction mixture. Virus in the medium was passaged on C6-36 cells to produce stocks, and these stocks had titers and plaque morphologies similar to those of the parental dengue virus type 2. Intracellular dengue virus RNA from cells infected with transcript-derived virus contained an introduced BstEII site, proving that infectivity was derived from RNA transcripts and not from contamination with parental dengue virus. Transcript-derived virus was comparable to dengue virus type 2 for growth and protein expression in tissue culture cells. Sequence analysis of the dengue virus cDNA in one full-length clone revealed only one unexpected silent mutation. By using yeast technology, it will be easy to introduce specific mutations into the dengue virus cDNA, allowing analysis of the virus phenotype in cells transfected with mutant transcripts. PMID:9188607

  15. Physical properties of naked DNA influence nucleosome positioning and correlate with transcription start and termination sites in yeast

    PubMed Central

    2011-01-01

    Background In eukaryotic organisms, DNA is packaged into chromatin structure, where most of DNA is wrapped into nucleosomes. DNA compaction and nucleosome positioning have clear functional implications, since they modulate the accessibility of genomic regions to regulatory proteins. Despite the intensive research effort focused in this area, the rules defining nucleosome positioning and the location of DNA regulatory regions still remain elusive. Results Naked (histone-free) and nucleosomal DNA from yeast were digested by microccocal nuclease (MNase) and sequenced genome-wide. MNase cutting preferences were determined for both naked and nucleosomal DNAs. Integration of their sequencing profiles with DNA conformational descriptors derived from atomistic molecular dynamic simulations enabled us to extract the physical properties of DNA on a genomic scale and to correlate them with chromatin structure and gene regulation. The local structure of DNA around regulatory regions was found to be unusually flexible and to display a unique pattern of nucleosome positioning. Ab initio physical descriptors derived from molecular dynamics were used to develop a computational method that accurately predicts nucleosome enriched and depleted regions. Conclusions Our experimental and computational analyses jointly demonstrate a clear correlation between sequence-dependent physical properties of naked DNA and regulatory signals in the chromatin structure. These results demonstrate that nucleosome positioning around TSS (Transcription Start Site) and TTS (Transcription Termination Site) (at least in yeast) is strongly dependent on DNA physical properties, which can define a basal regulatory mechanism of gene expression. PMID:21981773

  16. Assessing genetic diversity among Brettanomyces yeasts by DNA fingerprinting and whole-genome sequencing.

    PubMed

    Crauwels, Sam; Zhu, Bo; Steensels, Jan; Busschaert, Pieter; De Samblanx, Gorik; Marchal, Kathleen; Willems, Kris A; Verstrepen, Kevin J; Lievens, Bart

    2014-07-01

    Brettanomyces yeasts, with the species Brettanomyces (Dekkera) bruxellensis being the most important one, are generally reported to be spoilage yeasts in the beer and wine industry due to the production of phenolic off flavors. However, B. bruxellensis is also known to be a beneficial contributor in certain fermentation processes, such as the production of certain specialty beers. Nevertheless, despite its economic importance, Brettanomyces yeasts remain poorly understood at the genetic and genomic levels. In this study, the genetic relationship between more than 50 Brettanomyces strains from all presently known species and from several sources was studied using a combination of DNA fingerprinting techniques. This revealed an intriguing correlation between the B. bruxellensis fingerprints and the respective isolation source. To further explore this relationship, we sequenced a (beneficial) beer isolate of B. bruxellensis (VIB X9085; ST05.12/22) and compared its genome sequence with the genome sequences of two wine spoilage strains (AWRI 1499 and CBS 2499). ST05.12/22 was found to be substantially different from both wine strains, especially at the level of single nucleotide polymorphisms (SNPs). In addition, there were major differences in the genome structures between the strains investigated, including the presence of large duplications and deletions. Gene content analysis revealed the presence of 20 genes which were present in both wine strains but absent in the beer strain, including many genes involved in carbon and nitrogen metabolism, and vice versa, no genes that were missing in both AWRI 1499 and CBS 2499 were found in ST05.12/22. Together, this study provides tools to discriminate Brettanomyces strains and provides a first glimpse at the genetic diversity and genome plasticity of B. bruxellensis. PMID:24814796

  17. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing

    PubMed Central

    Crauwels, Sam; Zhu, Bo; Steensels, Jan; Busschaert, Pieter; De Samblanx, Gorik; Marchal, Kathleen; Willems, Kris A.

    2014-01-01

    Brettanomyces yeasts, with the species Brettanomyces (Dekkera) bruxellensis being the most important one, are generally reported to be spoilage yeasts in the beer and wine industry due to the production of phenolic off flavors. However, B. bruxellensis is also known to be a beneficial contributor in certain fermentation processes, such as the production of certain specialty beers. Nevertheless, despite its economic importance, Brettanomyces yeasts remain poorly understood at the genetic and genomic levels. In this study, the genetic relationship between more than 50 Brettanomyces strains from all presently known species and from several sources was studied using a combination of DNA fingerprinting techniques. This revealed an intriguing correlation between the B. bruxellensis fingerprints and the respective isolation source. To further explore this relationship, we sequenced a (beneficial) beer isolate of B. bruxellensis (VIB X9085; ST05.12/22) and compared its genome sequence with the genome sequences of two wine spoilage strains (AWRI 1499 and CBS 2499). ST05.12/22 was found to be substantially different from both wine strains, especially at the level of single nucleotide polymorphisms (SNPs). In addition, there were major differences in the genome structures between the strains investigated, including the presence of large duplications and deletions. Gene content analysis revealed the presence of 20 genes which were present in both wine strains but absent in the beer strain, including many genes involved in carbon and nitrogen metabolism, and vice versa, no genes that were missing in both AWRI 1499 and CBS 2499 were found in ST05.12/22. Together, this study provides tools to discriminate Brettanomyces strains and provides a first glimpse at the genetic diversity and genome plasticity of B. bruxellensis. PMID:24814796

  18. Human DNA-Damage-Inducible 2 Protein Is Structurally and Functionally Distinct from Its Yeast Ortholog

    PubMed Central

    Sivá, Monika; Svoboda, Michal; Veverka, Václav; Trempe, Jean-François; Hofmann, Kay; Kožíšek, Milan; Hexnerová, Rozálie; Sedlák, František; Belza, Jan; Brynda, Jiří; Šácha, Pavel; Hubálek, Martin; Starková, Jana; Flaisigová, Iva; Konvalinka, Jan; Šašková, Klára Grantz

    2016-01-01

    Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor. All Ddi1-like family members also contain a highly conserved retroviral protease-like (RVP) domain with unknown substrate specificity. While the structure and biological function of yeast Ddi1 have been investigated, no such analysis is available for the human homologs. To address this, we solved the 3D structures of the human Ddi2 UBL and RVP domains and identified a new helical domain that extends on either side of the RVP dimer. While Ddi1-like proteins from all vertebrates lack a UBA domain, we identify a novel ubiquitin-interacting motif (UIM) located at the C-terminus of the protein. The UIM showed a weak yet specific affinity towards ubiquitin, as did the Ddi2 UBL domain. However, the full-length Ddi2 protein is unable to bind to di-ubiquitin chains. While proteomic analysis revealed no activity, implying that the protease requires other factors for activation, our structural characterization of all domains of human Ddi2 sets the stage for further characterization. PMID:27461074

  19. Human DNA-Damage-Inducible 2 Protein Is Structurally and Functionally Distinct from Its Yeast Ortholog.

    PubMed

    Sivá, Monika; Svoboda, Michal; Veverka, Václav; Trempe, Jean-François; Hofmann, Kay; Kožíšek, Milan; Hexnerová, Rozálie; Sedlák, František; Belza, Jan; Brynda, Jiří; Šácha, Pavel; Hubálek, Martin; Starková, Jana; Flaisigová, Iva; Konvalinka, Jan; Šašková, Klára Grantz

    2016-01-01

    Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor. All Ddi1-like family members also contain a highly conserved retroviral protease-like (RVP) domain with unknown substrate specificity. While the structure and biological function of yeast Ddi1 have been investigated, no such analysis is available for the human homologs. To address this, we solved the 3D structures of the human Ddi2 UBL and RVP domains and identified a new helical domain that extends on either side of the RVP dimer. While Ddi1-like proteins from all vertebrates lack a UBA domain, we identify a novel ubiquitin-interacting motif (UIM) located at the C-terminus of the protein. The UIM showed a weak yet specific affinity towards ubiquitin, as did the Ddi2 UBL domain. However, the full-length Ddi2 protein is unable to bind to di-ubiquitin chains. While proteomic analysis revealed no activity, implying that the protease requires other factors for activation, our structural characterization of all domains of human Ddi2 sets the stage for further characterization. PMID:27461074

  20. Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments.

    PubMed

    Dmytruk, Kostyantyn V; Voronovsky, Andriy Y; Sibirny, Andriy A

    2006-09-01

    The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata. PMID:16770625

  1. Coordination of DNA damage tolerance mechanisms with cell cycle progression in fission yeast

    PubMed Central

    Callegari, A. John; Kelly, Thomas J.

    2016-01-01

    ABSTRACT DNA damage tolerance (DDT) mechanisms allow cells to synthesize a new DNA strand when the template is damaged. Many mutations resulting from DNA damage in eukaryotes are generated during DDT when cells use the mutagenic translesion polymerases, Rev1 and Polζ, rather than mechanisms with higher fidelity. The coordination among DDT mechanisms is not well understood. We used live-cell imaging to study the function of DDT mechanisms throughout the cell cycle of the fission yeast Schizosaccharomyces pombe. We report that checkpoint-dependent mitotic delay provides a cellular mechanism to ensure the completion of high fidelity DDT, largely by homology-directed repair (HDR). DDT by mutagenic polymerases is suppressed during the checkpoint delay by a mechanism dependent on Rad51 recombinase. When cells pass the G2/M checkpoint and can no longer delay mitosis, they completely lose the capacity for HDR and simultaneously exhibit a requirement for Rev1 and Polζ. Thus, DDT is coordinated with the checkpoint response so that the activity of mutagenic polymerases is confined to a vulnerable period of the cell cycle when checkpoint delay and HDR are not possible. PMID:26652183

  2. Quantitative epigenetics through epigenomic perturbation of isogenic lines.

    PubMed

    Johannes, Frank; Colomé-Tatché, Maria

    2011-05-01

    Interindividual differences in chromatin states at a locus (epialleles) can result in gene expression changes that are sometimes transmitted across generations. In this way, they can contribute to heritable phenotypic variation in natural and experimental populations independent of DNA sequence. Recent molecular evidence shows that epialleles often display high levels of transgenerational instability. This property gives rise to a dynamic dimension in phenotypic inheritance. To be able to incorporate these non-Mendelian features into quantitative genetic models, it is necessary to study the induction and the transgenerational behavior of epialleles in controlled settings. Here we outline a general experimental approach for achieving this using crosses of epigenomically perturbed isogenic lines in mammalian and plant species. We develop a theoretical description of such crosses and model the relationship between epiallelic instability, recombination, parent-of-origin effects, as well as transgressive segregation and their joint impact on phenotypic variation across generations. In the limiting case of fully stable epialleles our approach reduces to the classical theory of experimental line crosses and thus illustrates a fundamental continuity between genetic and epigenetic inheritance. We consider data from a panel of Arabidopsis epigenetic recombinant inbred lines and explore estimates of the number of quantitative trait loci for plant height that resulted from a manipulation of DNA methylation levels in one of the two isogenic founder strains. PMID:21385727

  3. The C-Terminal Domain of Yeast PCNA Is Required for Physical And Functional Interactions With Cdc9 DNA Ligase

    SciTech Connect

    Vijayakumar, S.; Chapados, B.R.; Schmidt, K.H.; Kolodner, R.D.; Tainer, J.A.; Tomkinson, A.E.

    2007-07-13

    There is compelling evidence that proliferating cell nuclear antigen (PCNA), a DNA sliding clamp, co-ordinates the processing and joining of Okazaki fragments during eukaryotic DNA replication. However, a detailed mechanistic understanding of functional PCNA:ligase I interactions has been incomplete. Here we present the co-crystal structure of yeast PCNA with a peptide encompassing the conserved PCNA interaction motif of Cdc9, yeast DNA ligase I. The Cdc9 peptide contacts both the inter-domain connector loop (IDCL) and residues near the C-terminus of PCNA. Complementary mutational and biochemical results demonstrate that these two interaction interfaces are required for complex formation both in the absence of DNA and when PCNA is topologically linked to DNA. Similar to the functionally homologous human proteins, yeast RFC interacts with and inhibits Cdc9 DNA ligase whereas the addition of PCNA alleviates inhibition by RFC. Here we show that the ability of PCNA to overcome RFC-mediated inhibition of Cdc9 is dependent upon both the IDCL and the C-terminal interaction interfaces of PCNA. Together these results demonstrate the functional significance of the {beta}-zipper structure formed between the C-terminal domain of PCNA and Cdc9 and reveal differences in the interactions of FEN-1 and Cdc9 with the two PCNA interfaces that may contribute to the coordinated, sequential action of these enzymes.

  4. Expression cloning in yeast of a cDNA encoding a broad specificity amino acid permease from Arabidopsis thaliana.

    PubMed Central

    Frommer, W B; Hummel, S; Riesmeier, J W

    1993-01-01

    To study amino acid transport in plants at the molecular level, we have isolated an amino acid permease cDNA from Arabidopsis thaliana by complementation of a yeast mutant defective in proline uptake with a cDNA. The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows no significant homologies to other known transporters. Expression of the cDNA enables the yeast mutant to take up L-[14C]proline. Competition studies argue for a broad but stereospecific substrate recognition by the permease, which resembles neutral or general amino acid transport systems from Chlorella and higher plants. Both pH dependence and inhibition by protonophores are consistent with a proton symport mechanism. Images Fig. 1 PMID:8327465

  5. Replication of single-stranded DNA templates by primase-polymerase complexes of the yeast, Saccharomyces cerevisiae.

    PubMed Central

    Biswas, E E; Biswas, S B

    1988-01-01

    A partially purified primase-polymerase complex from the yeast, Saccharomyces cerevisiae, was capable of replicating a single stranded circular phage DNA into a replicative form with high efficiency. The primase-polymerase complex exhibited primase activity and polymerase activity on singly primed circular ssDNA as well as on gapped DNA. In addition, it was able to replicate an unprimed, single-stranded, circular phage DNA through a coupled primase-polymerase action. On Biogel A-O.5m filtration the primase-polymerase activities appeared in the void volume, demonstrating a mass of greater than 500 kilodaltons. Primase and various primase-polymerase complexes synthesized unique primers on single stranded DNA templates and the size distribution of primers was dependent on the structure of the DNA and the nature of the primase-polymerase assembly. Images PMID:3041377

  6. A role for DNA polymerase alpha in epigenetic control of transcriptional silencing in fission yeast.

    PubMed

    Nakayama Ji; Allshire, R C; Klar, A J; Grewal, S I

    2001-06-01

    In the fission yeast Schizosaccharomyces pombe, transcriptional silencing at the mating-type region, centromeres and telomeres is epigenetically controlled, and results from the assembly of higher order chromatin structures. Chromatin proteins associated with these silenced loci are believed to serve as molecular bookmarks that help promote inheritance of the silenced state during cell division. Specifically, a chromodomain protein Swi6 is believed to be an important determinant of the epigenetic imprint. Here, we show that a mutation in DNA polymerase alpha (pol(alpha)) affects Swi6 localization at the mating-type region and causes a 45-fold increase in spontaneous transition from the silenced epigenetic state to the expressed state. We also demonstrate that pol(alpha) mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polalpha and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Pol(alpha) in vitro. Moreover, silencing-defective mutant Pol(alpha) displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Pol(alpha), in heterochromatin assembly and inheritance of epigenetic chromatin structures. PMID:11387218

  7. Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks.

    PubMed

    Willis, Nicholas A; Zhou, Chunshui; Elia, Andrew E H; Murray, Johanne M; Carr, Antony M; Elledge, Stephen J; Rhind, Nicholas

    2016-06-28

    The cellular response to DNA damage during S-phase regulates a complicated network of processes, including cell-cycle progression, gene expression, DNA replication kinetics, and DNA repair. In fission yeast, this S-phase DNA damage response (DDR) is coordinated by two protein kinases: Rad3, the ortholog of mammalian ATR, and Cds1, the ortholog of mammalian Chk2. Although several critical downstream targets of Rad3 and Cds1 have been identified, most of their presumed targets are unknown, including the targets responsible for regulating replication kinetics and coordinating replication and repair. To characterize targets of the S-phase DDR, we identified proteins phosphorylated in response to methyl methanesulfonate (MMS)-induced S-phase DNA damage in wild-type, rad3∆, and cds1∆ cells by proteome-wide mass spectrometry. We found a broad range of S-phase-specific DDR targets involved in gene expression, stress response, regulation of mitosis and cytokinesis, and DNA replication and repair. These targets are highly enriched for proteins required for viability in response to MMS, indicating their biological significance. Furthermore, the regulation of these proteins is similar in fission and budding yeast, across 300 My of evolution, demonstrating a deep conservation of S-phase DDR targets and suggesting that these targets may be critical for maintaining genome stability in response to S-phase DNA damage across eukaryotes. PMID:27298342

  8. Use of yeast nuclear DNA sequences to define the mitochondrial RNA polymerase promoter in vitro.

    PubMed Central

    Marczynski, G T; Schultz, P W; Jaehning, J A

    1989-01-01

    We have extended an earlier observation that the TATA box for the nuclear GAL10 gene serves as a promoter for the mitochondrial RNA polymerase in in vitro transcription reactions (C. S. Winkley, M. J. Keller, and J. A. Jaehning, J. Biol. Chem. 260:14214-14223, 1985). In this work, we demonstrate that other nuclear genes also have upstream sequences that function in vitro as mitochondrial RNA polymerase promoters. These genes include the GAL7 and MEL1 genes, which are regulated in concert with the GAL10 gene, the sigma repetitive element, and the 2 microns plasmid origin of replication. We used in vitro transcription reactions to test a large number of nuclear DNA sequences that contain critical mitochondrial promoter sequences as defined by Biswas et al. (T. K. Biswas, J. C. Edwards, M. Rabinowitz, and G. S. Getz, J. Biol. Chem. 262:13690-13696, 1987). The results of these experiments allowed us to extend the definition of essential promoter elements. This extended sequence, -ACTATAAACGatcATAG-, was frequently found in the upstream regulatory regions of nuclear genes. On the basis of these observations, we hypothesized that either (i) a catalytic RNA polymerase related to the mitochondrial enzyme functions in the nucleus of the yeast cell or (ii) a DNA sequence recognition factor is shared by the two genetic compartments. By using cells deficient in the catalytic core of the mitochondrial RNA polymerase (rpo41-) and sensitive assays for transcripts initiating from the nuclear promoter sequences, we have conclusively ruled out a role for the catalytic RNA polymerase in synthesizing transcripts from all of the nuclear sequences analyzed. The possibility that a DNA sequence recognition factor functions in both the nucleus and the mitochondria remains to be tested. Images PMID:2677667

  9. Prevalence and Dynamics of Ribosomal DNA Micro-heterogeneity Are Linked to Population History in Two Contrasting Yeast Species

    PubMed Central

    James, Stephen A.; West, Claire; Davey, Robert P.; Dicks, Jo; Roberts, Ian N.

    2016-01-01

    Despite the considerable number and taxonomic breadth of past and current genome sequencing projects, many of which necessarily encompass the ribosomal DNA, detailed information on the prevalence and evolutionary significance of sequence variation in this ubiquitous genomic region are severely lacking. Here, we attempt to address this issue in two closely related yet contrasting yeast species, the baker’s yeast Saccharomyces cerevisiae and the wild yeast Saccharomyces paradoxus. By drawing on existing datasets from the Saccharomyces Genome Resequencing Project, we identify a rich seam of ribosomal DNA sequence variation, characterising 1,068 and 970 polymorphisms in 34 S. cerevisiae and 26 S. paradoxus strains respectively. We discover the two species sets exhibit distinct mutational profiles. Furthermore, we show for the first time that unresolved rDNA sequence variation resulting from imperfect concerted evolution of the ribosomal DNA region follows a U-shaped allele frequency distribution in each species, similar to loci that evolve under non-concerted mechanisms but arising through rather different evolutionary processes. Finally, we link differences between the shapes of these allele frequency distributions to the two species’ contrasting population histories. PMID:27345953

  10. Prevalence and Dynamics of Ribosomal DNA Micro-heterogeneity Are Linked to Population History in Two Contrasting Yeast Species.

    PubMed

    James, Stephen A; West, Claire; Davey, Robert P; Dicks, Jo; Roberts, Ian N

    2016-01-01

    Despite the considerable number and taxonomic breadth of past and current genome sequencing projects, many of which necessarily encompass the ribosomal DNA, detailed information on the prevalence and evolutionary significance of sequence variation in this ubiquitous genomic region are severely lacking. Here, we attempt to address this issue in two closely related yet contrasting yeast species, the baker's yeast Saccharomyces cerevisiae and the wild yeast Saccharomyces paradoxus. By drawing on existing datasets from the Saccharomyces Genome Resequencing Project, we identify a rich seam of ribosomal DNA sequence variation, characterising 1,068 and 970 polymorphisms in 34 S. cerevisiae and 26 S. paradoxus strains respectively. We discover the two species sets exhibit distinct mutational profiles. Furthermore, we show for the first time that unresolved rDNA sequence variation resulting from imperfect concerted evolution of the ribosomal DNA region follows a U-shaped allele frequency distribution in each species, similar to loci that evolve under non-concerted mechanisms but arising through rather different evolutionary processes. Finally, we link differences between the shapes of these allele frequency distributions to the two species' contrasting population histories. PMID:27345953

  11. Yeast mitochondrial HMG proteins: DNA-binding properties of the most evolutionarily divergent component of mitochondrial nucleoids.

    PubMed

    Bakkaiova, Jana; Marini, Victoria; Willcox, Smaranda; Nosek, Jozef; Griffith, Jack D; Krejci, Lumir; Tomaska, Lubomir

    2016-01-01

    Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA in vivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species. PMID:26647378

  12. Yeast mitochondrial HMG proteins: DNA-binding properties of the most evolutionarily divergent component of mitochondrial nucleoids

    PubMed Central

    Bakkaiova, Jana; Marini, Victoria; Willcox, Smaranda; Nosek, Jozef; Griffith, Jack D.; Krejci, Lumir; Tomaska, Lubomir

    2015-01-01

    Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA in vivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species. PMID:26647378

  13. The expression of the open reading frame of "Arabidopsis" CAX1, but not its cDNA, confers metal tolerance in yeast

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The biochemical properties and regulation of several plant CAX (CAtion eXchanger)-type vacuolar Ca (2+)/H (+) exchangers have been extensively analyzed in yeast expression assays. In the present study, we compare and contrast the phenotypes of yeast cells expressing the CAX1 cDNA and open reading fr...

  14. A chimeric vacuolar Na(+)/H(+) antiporter gene evolved by DNA family shuffling confers increased salt tolerance in yeast.

    PubMed

    Wu, Guangxia; Wang, Gang; Ji, Jing; Li, Yong; Gao, Hailing; Wu, Jiang; Guan, Wenzhu

    2015-06-10

    The vacuolar Na(+)/H(+) antiporter plays an important role in maintaining ionic homeostasis and the osmotic balance of the cell with the environment by sequestering excessive cytoplasmic Na(+) into the vacuole. However, the relatively low Na(+)/H(+) exchange efficiency of the identified Na(+)/H(+) antiporter could limit its application in the molecular breeding of salt tolerant crops. In this study, DNA family shuffling was used to create chimeric Na(+)/H(+) antiporters with improved transport activity. Two homologous Na(+)/H(+) antiporters from halophytes Salicornia europaea (SeNHX1) and Suaeda salsa (SsNHX1) were shuffled to generate a diverse gene library. Using a high-throughput screening system of yeast complementation, a novel chimeric protein SseNHX1 carrying 12 crossover positions and 2 point mutations at amino acid level was selected. Expression of SseNHX1 in yeast mutant exhibited approximately 46% and 22% higher salt tolerance ability in yeast growth test than that of SsNHX1and SeNHX1, respectively. Measurements of the ion contents demonstrated that SseNHX1 protein in yeast cells accumulated more Na(+) and slightly more K(+) than the parental proteins did. Furthermore, this chimera also conferred increased tolerance to LiCl and a similar tolerance to hygromycin B compared with the parental proteins in yeast. PMID:25784157

  15. RAP1 stimulates single- to double-strand association of yeast telomeric DNA: implications for telomere-telomere interactions.

    PubMed Central

    Gilson, E; Müller, T; Sogo, J; Laroche, T; Gasser, S M

    1994-01-01

    Repressor Activator Protein 1 (RAP1) of Saccharomyces cerevisiae is an abundant nuclear protein implicated in telomere length maintenance, transactivation, and in the establishment of silent chromatin domains. The RAP1 binding site 5' of the yeast HIS4 gene is also a region of hyperrecombination in meiosis. We report here that as RAP1 binds its recognition consensus, it appears to untwist double-stranded DNA, which we detect as the introduction of a negative supercoil in circularization assays. Coincident with the RAP1-dependent untwisting, we observe stimulation of the association of a single-stranded yeast telomeric sequence with its homologous double-stranded sequence in a supercoiled plasmid. This unusual distortion of the DNA double helix by RAP1 may contribute to the RAP1-dependent enhancement of recombination rates and promote non-duplex strand interactions at telomeres. Images PMID:7816621

  16. HRR25, a putative protein kinase from budding yeast: Association with repair of damaged DNA

    SciTech Connect

    Hoekstra, M.F.; Ou, A.C.; DeMaggio, A.J.; Burbee, D.G. ); Liskay, R.M. ); Heffron, F. )

    1991-08-30

    In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH{sub 2}-terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.

  17. Cloning yeast actin cDNA leads to an investigative approach for the molecular biology laboratory.

    PubMed

    Black, Michael W; Tuan, Alice; Jonasson, Erin

    2008-05-01

    The emergence of molecular tools in multiple disciplines has elevated the importance of undergraduate laboratory courses that train students in molecular biology techniques. Although it would also be desirable to provide students with opportunities to apply these techniques in an investigative manner, this is generally not possible in the classroom because of the preparation, expense, and logistics involved in independent student projects. The authors have designed a 10-week lab series that mimics the research environment by tying separate fundamental lab techniques to a common goal: to build a plasmid with yeast actin cDNA cloned in a particular orientation. In the process of completing this goal, a problem arises in that students are unable to obtain the target plasmid and instead only recover the gene cloned in the opposite orientation. To address this problem, students identify four plausible hypotheses and work in teams to address them by designing and executing experiments. This project reinforces the utility and flexibility of techniques covered earlier in the class and serves to develop their skills in experimental design and analysis. As the project is focused on one problem, the diversity of experimental approaches is limited and may be prepared in advance with little additional expense in reagents or technical support. PMID:21591194

  18. Identification of barriers to rotation of DNA segments in yeast from the topology of DNA rings excised by an inducible site-specific recombinase.

    PubMed Central

    Gartenberg, M R; Wang, J C

    1993-01-01

    Controlled excision of DNA segments to yield intracellular DNA rings of well-defined sequences was utilized to study the determinants of transcriptional supercoiling of closed circular DNA in the yeast Saccharomyces cerevisiae. In delta top1 top2ts strains of S. cerevisiae expressing Escherichia coli DNA topoisomerase I, accumulation of positive supercoils in intracellular DNA normally occurs upon thermal inactivation of DNA topoisomerase II because of the simultaneous generation of positively and negatively supercoiled domains by transcription and the preferential relaxation of the latter by the bacterial enzyme. Positive supercoil accumulation in DNA rings is shown to depend on the presence of specific sequence elements; one likely cause of this dependence is that the persistence of oppositely supercoiled domains in an intracellular DNA ring requires the presence of barriers to rotation of the DNA segments connecting the domains. Analysis of the S. cerevisiae 2-microns plasmid partition system by this approach suggests that the plasmid-encoded REP1 and REP2 proteins are involved in forming such a barrier in DNA containing the REP3 sequence. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8248138

  19. RNA Polymerase I and Fob1 contributions to transcriptional silencing at the yeast rDNA locus.

    PubMed

    Buck, Stephen W; Maqani, Nazif; Matecic, Mirela; Hontz, Robert D; Fine, Ryan D; Li, Mingguang; Smith, Jeffrey S

    2016-07-27

    RNA polymerase II (Pol II)-transcribed genes embedded within the yeast rDNA locus are repressed through a Sir2-dependent process called 'rDNA silencing'. Sir2 is recruited to the rDNA promoter through interactions with RNA polymerase I (Pol I), and to a pair of DNA replication fork block sites (Ter1 and Ter2) through interaction with Fob1. We utilized a reporter gene (mURA3) integrated adjacent to the leftmost rDNA gene to investigate localized Pol I and Fob1 functions in silencing. Silencing was attenuated by loss of Pol I subunits or insertion of an ectopic Pol I terminator within the adjacent rDNA gene. Silencing left of the rDNA array is naturally attenuated by the presence of only one intact Fob1 binding site (Ter2). Repair of the 2nd Fob1 binding site (Ter1) dramatically strengthens silencing such that it is no longer impacted by local Pol I transcription defects. Global loss of Pol I activity, however, negatively affects Fob1 association with the rDNA. Loss of Ter2 almost completely eliminates localized silencing, but is restored by artificially targeting Fob1 or Sir2 as Gal4 DNA binding domain fusions. We conclude that Fob1 and Pol I make independent contributions to establishment of silencing, though Pol I also reinforces Fob1-dependent silencing. PMID:27060141

  20. Analysis of repair and mutagenesis of chromium-induced DNA damage in yeast, mammalian cells, and transgenic mice.

    PubMed Central

    Cheng, L; Liu, S; Dixon, K

    1998-01-01

    Chromium (Cr) is a widespread environmental contaminant and a known human carcinogen. We have used shuttle vector systems in yeast, mammalian cells, and transgenic mice to characterize the mutational specificity and premutational DNA damage induced by Cr(VI) and its reduction intermediates in order to elucidate the mechanism by which Cr induces mutations. In the yeast system, treatment of vector-containing cells with Cr(VI) results in a dose-dependent increase in mutations in the SUP4-o target gene of the vector; mutagenesis is enhanced in an apn-1 yeast mutant, deficient in the capacity to repair oxidative-type DNA damage. In vector-containing mammalian cells, treatment with Cr(VI) also results in a dose-dependent increase in mutations in the vector target gene supF. The Cr-induced mutations in supF occurred mostly at G:C base pairs and were widely distributed across the gene, a pattern similar to those observed with ionizing radiation or hydrogen peroxide. These results support the hypothesis that Cr(VI)-induced oxidative-type DNA damage is responsible for Cr mutagenesis in the cell. Recently these studies were extended into the Big Blue transgenic mouse system in which Cr-induced mutagenesis was observed in the lung, the target organ for Cr carcinogenesis in humans. Analysis of the spectrum of these mutations will test whether Cr mutagenesis occurs by similar mechanisms in the intact animal as in cell culture systems and yeast. Images Figure 2 Figure 3 PMID:9703488

  1. Shared Genetic Pathways Contribute to the Tolerance of Endogenous and Low-Dose Exogenous DNA Damage in Yeast

    PubMed Central

    Lehner, Kevin; Jinks-Robertson, Sue

    2014-01-01

    DNA damage that escapes repair and blocks replicative DNA polymerases is tolerated by bypass mechanisms that fall into two general categories: error-free template switching and error-prone translesion synthesis. Prior studies of DNA damage responses in Saccharomyces cerevisiae have demonstrated that repair mechanisms are critical for survival when a single, high dose of DNA damage is delivered, while bypass/tolerance mechanisms are more important for survival when the damage level is low and continuous (acute and chronic damage, respectively). In the current study, epistatic interactions between DNA-damage tolerance genes were examined and compared when haploid yeast cells were exposed to either chronic ultraviolet light or chronic methyl methanesulfonate. Results demonstrate that genes assigned to error-free and error-prone bypass pathways similarly promote survival in the presence of each type of chronic damage. In addition to using defined sources of chronic damage, rates of spontaneous mutations generated by the Pol ζ translesion synthesis DNA polymerase (complex insertions in a frameshift-reversion assay) were used to infer epistatic interactions between the same genes. Similar epistatic interactions were observed in analyses of spontaneous mutation rates, suggesting that chronic DNA-damage responses accurately reflect those used to tolerate spontaneous lesions. These results have important implications when considering what constitutes a safe and acceptable level of exogenous DNA damage. PMID:25060101

  2. DNA-dependent DNA polymerase from yeast mitochondria. Dependence of enzyme activity on conditions of cell growth, and properties of the highly purified polymerase.

    PubMed

    Wintersberger, U; Blutsch, H

    1976-09-01

    The activity of DNA polymerase was determined in gradient-purified mitochondria from yeast cells grown under a variety of conditions. The specific enzyme activity was found to be dependent on the degree of aeration of the cells, and on the carbon source used for the medium. It was sensitive to glucose repression, and was enhanced about two-fold by the growth of yeast cells in the presence of ethidium bromide. Mitochondria DNA polymerase was highly purified and several properties were determined. Sucrose density gradient centrifugation, and dodecylsulfate-polyacylamide gel electrophoresis revealed the following structure: a monomer of molecular weight around 60 000 aggregated under relatively high salt concentration (0.2 M phosphate buffer) to a dimer of about 120 000 which under low salt concentration (0.2 M Tris-HCl buffer) formed higher aggregates. For optimal activity an Mg2+ ion concentration of 50 mM was found necessary, Mn ions did not promote activity at any concentration tested (0.5--50 mM). Indeed, if added to Mg2+-containing assays, Mn2+ strongly inhibited enzyme activity at low concentrations. This might be an explanation for the inducation of mitochondrial mutants in yeast cells grown in the presence of Mn2+ ions. Mitochondrial DNA polymerase activity was strongly inhibited by low concentrations of the -SH reagent p-chloromercuribenzoate, the nucleotide analogue cytosine arabinoside triphosphate also exerted an inhibitory effect. An about 50% decrease of activity was observed in the presence of 1 mM o-phenanthroline in assay mixture containing DNA at about the Km concentration. The enzyme preferred a gapped template primer, poly(dA) - (dT)10, over nicked DNA and was unable to use a polyribonucleotide template, poly(rA) - (dT)10. In the purest preparations no exonuclease activity could be detected. PMID:786635

  3. DNA Replication Forks Pause at Silent Origins near the HML Locus in Budding Yeast

    PubMed Central

    Wang, Yangzhou; Vujcic, Marija; Kowalski, David

    2001-01-01

    Chromosomal replicators in budding yeast contain an autonomously replicating sequence (ARS) that functions in a plasmid, but certain ARSs are silent as replication origins in their natural chromosomal context. In chromosome III, the HML ARS cluster (ARS302-ARS303-ARS320) and ARS301 flank the transcriptionally silent mating-type locus HML, and all of these ARSs are silent as replication origins. ARS301 and ARS302 function in transcriptional silencing mediated by the origin recognition complex (ORC) and a heterochromatin structure, while the functions of ARS303 and ARS320 are not known. In this work, we discovered replication fork pause sites at the HML ARS cluster and ARS301 by analyzing DNA replication intermediates from the chromosome via two-dimensional gel electrophoresis. The replication fork pause at the HML ARS cluster was independent of cis- and trans-acting mutations that abrogate transcriptional silencing at HML. Deletion of the HML ARS cluster led to loss of the pause site. Insertion of a single, heterologous ARS (ARS305) in place of the HML ARS cluster reconstituted the pause site, as did multiple copies of DNA elements (A and B1) that bind ORC. The orc2-1 mutation, known to alter replication timing at origins, did not detectably affect the pause but activated the silent origin at the HML ARS cluster in a minority of cells. Delaying the time of fork arrival at HML led to the elimination of the pause sites at the HML ARS cluster and at the copy of ARS305 inserted in place of the cluster. Loss of the pause sites was accompanied by activation of the silent origins in the majority of cells. Thus, replication fork movement near HML pauses at a silent origin which is competent for replication initiation but kept silent through Orc2p, a component of the replication initiator. Possible functions for replication fork pause sites in checkpoints, S-phase regulation, mating-type switching, and transcriptionally silent heterochromatin are discussed. PMID:11438651

  4. Isolation and DNA-binding characteristics of a protein involved in transcription activation of two divergently transcribed, essential yeast genes.

    PubMed Central

    Halfter, H; Müller, U; Winnacker, E L; Gallwitz, D

    1989-01-01

    We have identified a protein, BAF1, which has two oppositely oriented, partially overlapping binding sites within a symmetrical sequence located midway between and upstream of the divergently transcribed YPT1 and TUB2 genes of the yeast Saccharomyces cerevisiae. The 120 kd BAF1 protein was purified to near homogeneity and used to delineate the two binding sites and to identify apparent protein contact sites by the missing contact technique, methylation interference and by site-directed mutagenesis. The BAF1-recognition sequence contains a conserved TCN7ACG element recently identified at autonomously replicating sequences (ARS) and in the 5' and 3' flanking region of other yeast genes. The symmetrical sequence of the YPT1/TUB2 intergene region seems not to be involved in DNA replication but activates transcription in an orientation-independent fashion. Images PMID:2684633

  5. Chl1p, a DNA helicase-like protein in budding yeast, functions in sister-chromatid cohesion.

    PubMed Central

    Skibbens, Robert V

    2004-01-01

    From the time of DNA replication until anaphase onset, sister chromatids remain tightly paired along their length. Ctf7p/Eco1p is essential to establish sister-chromatid pairing during S-phase and associates with DNA replication components. DNA helicases precede the DNA replication fork and thus will first encounter chromatin sites destined for cohesion. In this study, I provide the first evidence that a DNA helicase is required for proper sister-chromatid cohesion. Characterizations of chl1 mutant cells reveal that CHL1 interacts genetically with both CTF7/ECO1 and CTF18/CHL12, two genes that function in sister-chromatid cohesion. Consistent with genetic interactions, Chl1p physically associates with Ctf7p/Eco1p both in vivo and in vitro. Finally, a functional assay reveals that Chl1p is critical for sister-chromatid cohesion. Within the budding yeast genome, Chl1p exhibits the highest degree of sequence similarity to human CHL1 isoforms and BACH1. Previous studies revealed that human CHLR1 exhibits DNA helicase-like activities and that BACH1 is a helicase-like protein that associates with the tumor suppressor BRCA1 to maintain genome integrity. Our findings document a novel role for Chl1p in sister-chromatid cohesion and provide new insights into the possible mechanisms through which DNA helicases may contribute to cancer progression when mutated. PMID:15020404

  6. Molecular visualization of the yeast Dmc1 protein ring and Dmc1-ssDNA nucleoprotein complex.

    PubMed

    Chang, Yuan-Chih; Lo, Yu-Hui; Lee, Ming-Hui; Leng, Chih-Hsiang; Hu, Su-Ming; Chang, Chia-Seng; Wang, Ting-Fang

    2005-04-26

    Saccharomyces cerevisiae Dmc1, a meiosis-specific homologue of RecA, catalyzes homologous pairing and strand exchange during meiotic DNA recombination. The purified budding yeast Dmc1 (ScDmc1) protein exhibits much weaker recombinase activity in vitro as compared to that of the Escherichia coli RecA protein. Using atomic force microscopy (AFM) with carbon nanotube tips, we found ScDmc1 forms rings with an external diameter of 18 nm and a central cavity of 4 nm. In the presence of single-stranded DNA (ssDNA), the majority of the ScDmc1 protein (90%) bound DNA as protein rings; only a small faction (10%) was able to form filamentous structure. In contrast, nearly all RecA proteins form fine helical nucleoprotein filaments with ssDNA under identical conditions. RecA-mediated recombinase activity is initiated through the nucleation of RecA onto ssDNA to form helical nucleoprotein filaments. Our results support the notion that ScDmc1 becomes catalytically active only when it forms a helical nucleoprotein filament with ssDNA. PMID:15835894

  7. The EGD1 product, a yeast homolog of human BTF3, may be involved in GAL4 DNA binding.

    PubMed Central

    Parthun, M R; Mangus, D A; Jaehning, J A

    1992-01-01

    A variety of techniques, including filter binding, footprinting, and gel retardation, can be used to assay the transcriptional activator GAL4 (Gal4p) through the initial steps of its purification from yeast cells. Following DNA affinity chromatography, Gal4p still bound DNA selectively when assayed by filter binding or footprinting. However, the affinity-purified protein was no longer capable of forming a stable complex with DNA, as assayed by gel retardation. Mixing the purified Gal4p with the flowthrough fraction from the DNA affinity column restored gel retardation complex formation. Gel retardation assays were used to monitor the purification of a heat-stable Gal4p-DNA complex stabilization activity from the affinity column flowthrough. The activity coeluted from the final purification step with polypeptides of 21 and 27 kDa. The yeast gene encoding the 21-kDa protein was cloned on the basis of its N-terminal amino acid sequence. The gene, named EGD1 (enhancer of GAL4 DNA binding), encodes a highly basic protein (21% lysine and arginine) with a predicted molecular mass of 16.5 kDa. The amino acid sequence of the EGD1 product, Egd1p, is highly similar to that of the human protein BTF3 (X. M. Zheng, D. Black, P. Chambon, and J. M. Egly, Nature [London] 344:556-559, 1990). Although an egd1 null mutant was viable and Gal+, induction of the galactose-regulated genes in the egd1 mutant strain was significantly reduced when cells were shifted from glucose to galactose. Images PMID:1448098

  8. A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast.

    PubMed

    Natsume, Toyoaki; Tsutsui, Yasuhiro; Sutani, Takashi; Dunleavy, Elaine M; Pidoux, Alison L; Iwasaki, Hiroshi; Shirahige, Katsuhiko; Allshire, Robin C; Yamao, Fumiaki

    2008-01-01

    Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication. PMID:18493607

  9. Proteome-wide Identification of Novel Ceramide-binding Proteins by Yeast Surface cDNA Display and Deep Sequencing.

    PubMed

    Bidlingmaier, Scott; Ha, Kevin; Lee, Nam-Kyung; Su, Yang; Liu, Bin

    2016-04-01

    Although the bioactive sphingolipid ceramide is an important cell signaling molecule, relatively few direct ceramide-interacting proteins are known. We used an approach combining yeast surface cDNA display and deep sequencing technology to identify novel proteins binding directly to ceramide. We identified 234 candidate ceramide-binding protein fragments and validated binding for 20. Most (17) bound selectively to ceramide, although a few (3) bound to other lipids as well. Several novel ceramide-binding domains were discovered, including the EF-hand calcium-binding motif, the heat shock chaperonin-binding motif STI1, the SCP2 sterol-binding domain, and the tetratricopeptide repeat region motif. Interestingly, four of the verified ceramide-binding proteins (HPCA, HPCAL1, NCS1, and VSNL1) and an additional three candidate ceramide-binding proteins (NCALD, HPCAL4, and KCNIP3) belong to the neuronal calcium sensor family of EF hand-containing proteins. We used mutagenesis to map the ceramide-binding site in HPCA and to create a mutant HPCA that does not bind to ceramide. We demonstrated selective binding to ceramide by mammalian cell-produced wild type but not mutant HPCA. Intriguingly, we also identified a fragment from prostaglandin D2synthase that binds preferentially to ceramide 1-phosphate. The wide variety of proteins and domains capable of binding to ceramide suggests that many of the signaling functions of ceramide may be regulated by direct binding to these proteins. Based on the deep sequencing data, we estimate that our yeast surface cDNA display library covers ∼60% of the human proteome and our selection/deep sequencing protocol can identify target-interacting protein fragments that are present at extremely low frequency in the starting library. Thus, the yeast surface cDNA display/deep sequencing approach is a rapid, comprehensive, and flexible method for the analysis of protein-ligand interactions, particularly for the study of non-protein ligands. PMID

  10. Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases.

    PubMed

    Saparbaev, M; Laval, J

    1994-06-21

    The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A.T to G.C transition. Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E. coli is associated with the 3-methyladenine DNA glycosylase II coded for by the alkA gene. This conclusion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, (ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E. coli alkA- mutant does not show any detectable hypoxanthine DNA glycosylase activity. Under the same experimental conditions, but using different substrates, the same amount of AlkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA. The Km for the latter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkylated DNA. Hypoxanthine is released as a free base during the reaction. Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T, G, C, and A were cleaved efficiently. ANPG protein, APDG protein, and MAG protein--the 3-methyladenine DNA glycosylases of human, rat, and yeast origin, respectively--were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested. PMID:8016081

  11. Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases.

    PubMed Central

    Saparbaev, M; Laval, J

    1994-01-01

    The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A.T to G.C transition. Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E. coli is associated with the 3-methyladenine DNA glycosylase II coded for by the alkA gene. This conclusion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, (ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E. coli alkA- mutant does not show any detectable hypoxanthine DNA glycosylase activity. Under the same experimental conditions, but using different substrates, the same amount of AlkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA. The Km for the latter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkylated DNA. Hypoxanthine is released as a free base during the reaction. Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T, G, C, and A were cleaved efficiently. ANPG protein, APDG protein, and MAG protein--the 3-methyladenine DNA glycosylases of human, rat, and yeast origin, respectively--were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested. Images PMID:8016081

  12. Complete DNA Sequence of Kuraishia capsulata Illustrates Novel Genomic Features among Budding Yeasts (Saccharomycotina)

    PubMed Central

    Morales, Lucia; Noel, Benjamin; Porcel, Betina; Marcet-Houben, Marina; Hullo, Marie-Francoise; Sacerdot, Christine; Tekaia, Fredj; Leh-Louis, Véronique; Despons, Laurence; Khanna, Varun; Aury, Jean-Marc; Barbe, Valérie; Couloux, Arnaud; Labadie, Karen; Pelletier, Eric; Souciet, Jean-Luc; Boekhout, Teun; Gabaldon, Toni; Wincker, Patrick; Dujon, Bernard

    2013-01-01

    The numerous yeast genome sequences presently available provide a rich source of information for functional as well as evolutionary genomics but unequally cover the large phylogenetic diversity of extant yeasts. We present here the complete sequence of the nuclear genome of the haploid-type strain of Kuraishia capsulata (CBS1993T), a nitrate-assimilating Saccharomycetales of uncertain taxonomy, isolated from tunnels of insect larvae underneath coniferous barks and characterized by its copious production of extracellular polysaccharides. The sequence is composed of seven scaffolds, one per chromosome, totaling 11.4 Mb and containing 6,029 protein-coding genes, ∼13.5% of which being interrupted by introns. This GC-rich yeast genome (45.7%) appears phylogenetically related with the few other nitrate-assimilating yeasts sequenced so far, Ogataea polymorpha, O. parapolymorpha, and Dekkera bruxellensis, with which it shares a very reduced number of tRNA genes, a novel tRNA sparing strategy, and a common nitrate assimilation cluster, three specific features to this group of yeasts. Centromeres were recognized in GC-poor troughs of each scaffold. The strain bears MAT alpha genes at a single MAT locus and presents a significant degree of conservation with Saccharomyces cerevisiae genes, suggesting that it can perform sexual cycles in nature, although genes involved in meiosis were not all recognized. The complete absence of conservation of synteny between K. capsulata and any other yeast genome described so far, including the three other nitrate-assimilating species, validates the interest of this species for long-range evolutionary genomic studies among Saccharomycotina yeasts. PMID:24317973

  13. Epidemiologic Study of Malassezia Yeasts in Acne Patients by Analysis of 26S rDNA PCR-RFLP

    PubMed Central

    Song, Young Chan; Hahn, Hyung Jin; Kim, Ji Young; Ko, Jong Hyun; Choe, Yong Beom; Ahn, Kyu Joong

    2011-01-01

    Background Although acne is a common follicular inflammatory dermatosis, studies of the relationship between Malassezia yeasts and acne have rarely been conducted. Objective We sought to identify Malassezia yeasts from acne patients and establish a relationship between specific types of species of Malassezia and acne. Methods Sixty acne patients were enrolled. Each strain obtained was identified as one of eleven species by 26S rDNA PCR-RFLP. We then compared these data with those of age- and sex-matched healthy subjects. Results Growth of Malassezia was evident in fewer patients with acne (50%) in comparison to controls (70.6%). M. restricta was dominant in patients with acne (23.9%), whereas M. globosa was most common (26.7%) in healthy controls. In the patients group, the rate was the highest (71.7%) in the twenties and, in terms of body site, the rate was the highest (60%) in the chest. In the control group, the rate was the highest (75.0%) in the thirties and in the forehead (85.0%). Conclusion The detection rate of Malassezia yeasts was conspicuously low in the acne patients group. Statistically significant differences were observed between the patient and the control groups in the twenties and thirties, and in terms of body site, in the forehead and chest. PMID:21909202

  14. Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen

    PubMed Central

    2011-01-01

    Background Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei. Findings Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates. Conclusions We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls. PMID:22088094

  15. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene.

    PubMed Central

    Kurtzman, C P; Robnett, C J

    1997-01-01

    Clinically important species of Candida and related organisms were compared for extent of nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region is sufficiently variable to allow reliable separation of all known clinically significant yeast species. Of the 204 described species examined, 21 appeared to be synonyms of previously described organisms. Phylogenetic relationships among the species are presented. PMID:9114410

  16. ORC-dependent and origin-specific initiation of DNA replication at defined foci in isolated yeast nuclei.

    PubMed

    Pasero, P; Braguglia, D; Gasser, S M

    1997-06-15

    We describe an in vitro replication assay from yeast in which the addition of intact nuclei to an S-phase nuclear extract results in the incorporation of deoxynucleotides into genomic DNA at spatially discrete foci. When BrdUTP is substituted for dTTP, part of the newly synthesized DNA shifts to a density on CsCl gradients, indicative of semiconservative replication. Initiation occurs in an origin-specific manner and can be detected in G1- or S-phase nuclei, but not in G2-phase or mitotic nuclei. The S-phase extract contains a heat- and 6-DMAP-sensitive component necessary to promote replication in G1-phase nuclei. Replication of nuclear DNA is blocked at the restrictive temperature in an orc2-1 mutant, and the inactive Orc2p cannot be complemented in trans by an extract containing wild-type ORC. The initiation of DNA replication in cln-deficient nuclei blocked in G1 indicates that the ORC-dependent prereplication complex is formed before Start. This represents the first nonviral and nonembryonic replication system in which DNA replication initiates in an ORC-dependent and origin-specific manner in vitro. PMID:9203578

  17. Reconstitution of a MEC1-independent checkpoint in yeast by expression of a novel human fork head cDNA.

    PubMed Central

    Pati, D; Keller, C; Groudine, M; Plon, S E

    1997-01-01

    A novel human cDNA, CHES1 (checkpoint suppressor 1), has been isolated by suppression of the mec1-1 checkpoint mutation in Saccharomyces cerevisiae. CHES1 suppresses a number of DNA damage-activated checkpoint mutations in S. cerevisiae, including mec1, rad9, rad24, dun1, and rad53. CHES1 suppression of sensitivity to DNA damage is specific for checkpoint-defective strains, in contrast to DNA repair-defective strains. Presence of CHES1 but not a control vector resulted in G2 delay after UV irradiation in checkpoint-defective strains, with kinetics, nuclear morphology, and cycloheximide resistance similar to those of a wild-type strain. CHES1 can also suppress the lethality, UV sensitivity, and G2 checkpoint defect of a mec1 null mutation. In contrast to this activity, CHES1 had no measurable effect on the replication checkpoint as assayed by hydroxyurea sensitivity of a mec1 strain. Sequence analysis demonstrates that CHES1 is a novel member of the fork head/Winged Helix family of transcription factors. Suppression of the checkpoint-defective phenotype requires a 200-amino-acid domain in the carboxy terminus of the protein which is distinct from the DNA binding site. Analysis of CHES1 activity is most consistent with activation of an alternative MEC1-independent checkpoint pathway in budding yeast. PMID:9154802

  18. Rapid generation of long tandem DNA repeat arrays by homologous recombination in yeast to study their function in mammalian genomes

    PubMed Central

    2011-01-01

    We describe here a method to rapidly convert any desirable DNA fragment, as small as 100 bp, into long tandem DNA arrays up to 140 kb in size that are inserted into a microbe vector. This method includes rolling-circle phi29 amplification (RCA) of the sequence in vitro and assembly of the RCA products in vivo by homologous recombination in the yeast Saccharomyces cerevisiae. The method was successfully used for a functional analysis of centromeric and pericentromeric repeats and construction of new vehicles for gene delivery to mammalian cells. The method may have general application in elucidating the role of tandem repeats in chromosome organization and dynamics. Each cycle of the protocol takes ~ two weeks to complete. PMID:21982381

  19. Activation of budding yeast replication origins and suppression of lethal DNA damage effects on origin function by ectopic expression of the co-chaperone protein Mge1.

    PubMed

    Trabold, Peter A; Weinberger, Martin; Feng, Li; Burhans, William C

    2005-04-01

    Initiation of DNA replication in eukaryotes requires the origin recognition complex (ORC) and other proteins that interact with DNA at origins of replication. In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with defects in initiation of DNA replication and in checkpoints that depend on DNA replication forks. Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC. A dosage suppressor screen identified the budding yeast co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug. Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation. In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin. Mge1p is the budding yeast homologue of the Escherichia coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences. Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA. PMID:15647270

  20. DNA polymerase alpha associated protein P1, a murine homolog of yeast MCM3, changes its intranuclear distribution during the DNA synthetic period.

    PubMed Central

    Kimura, H; Nozaki, N; Sugimoto, K

    1994-01-01

    We isolated a murine gene for the DNA polymerase alpha associated protein P1, which shares high homology with the budding yeast MCM3 protein, which is a member of a protein family involved in the early event of DNA replication having a putative DNA-dependent ATPase motif. Using a polyclonal anti-P1 antibody raised against a beta-galactosidase-P1 fusion protein, we identified at least two forms of P1 protein in the nucleus of a mouse cell line, an underphosphorylated form that was associated with a particular nuclear structure and a hyperphosphorylated form loosely bound to the nucleus. During progression through S phase, P1 disappeared, first from the euchromatic region, then from the heterochromatic region, apparently in parallel with temporally ordered DNA replication. Thus, it is likely that the underphosphorylated P1 is dissociated from the nuclear structure after DNA replication by cell cycle-dependent phosphorylation. This is the first direct observation of a protein whose behavior is consistent with that of a hypothetical factor which restricts the chromatin to replicate once per cell cycle in higher eukaryotes. Images PMID:7925275

  1. Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing

    PubMed Central

    Khade, Nilesh V.; Sugiyama, Tomohiko

    2016-01-01

    Yeast Rad52 (yRad52) has two important functions at homologous DNA recombination (HR); annealing complementary single-strand DNA (ssDNA) molecules and recruiting Rad51 recombinase onto ssDNA (recombination mediator activity). Its human homolog (hRAD52) has a lesser role in HR, and apparently lacks mediator activity. Here we show that yRad52 can load human Rad51 (hRAD51) onto ssDNA complexed with yeast RPA in vitro. This is biochemically equivalent to mediator activity because it depends on the C-terminal Rad51-binding region of yRad52 and on functional Rad52-RPA interaction. It has been reported that the N-terminal two thirds of both yRad52 and hRAD52 is essential for binding to and annealing ssDNA. Although a second DNA binding region has been found in the C-terminal region of yRad52, its role in ssDNA annealing is not clear. In this paper, we also show that the C-terminal region of yRad52, but not of hRAD52, is involved in ssDNA annealing. This suggests that the second DNA binding site is required for the efficient ssDNA annealing by yRad52. We propose an updated model of Rad52-mediated ssDNA annealing. PMID:27362509

  2. Trans-complementation by human apurinic endonuclease (Ape) of hypersensitivity to DNA damage and spontaneous mutator phenotype in apn1-yeast.

    PubMed Central

    Wilson, D M; Bennett, R A; Marquis, J C; Ansari, P; Demple, B

    1995-01-01

    Abasic (AP) sites in DNA are potentially lethal and mutagenic. 'Class II' AP endonucleases initiate the repair of these and other DNA lesions. In yeast, the predominant enzyme of this type is Apn1, and its elimination sensitizes the cells to killing by simple alkylating agents or oxidants, and raises the rate of spontaneous mutation. We investigated the ability of the major human class II AP endonuclease, Ape, which is structurally unrelated to Apn1, to replace the yeast enzyme in vivo. Confocal immunomicroscopy studies indicate that approximately 25% of the Ape expressed in yeast is present in the nucleus. High-level Ape expression corresponding to approximately 7000 molecules per nucleus, equal to the normal Apn1 copy number, restored resistance to methyl methanesulfonate to near wild-type levels in Apn1-deficient (apn1-) yeast. Ape expression in apn1- yeast provided little protection against H2O2 challenges, consistent with the weak 3'-repair diesterase activity of the human enzyme. Ape expression at approximately 2000 molecules per nucleus reduced the spontaneous mutation rate of apn1- yeast to that seen for wild-type cells. Because Ape has a powerful AP endonuclease but weak 3'-diesterase activity, these findings indicate that endogenously generated AP sites can drive spontaneous mutagenesis. Images PMID:8559661

  3. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins

    SciTech Connect

    Matviw, Yu, G.; Young, D. )

    1992-11-01

    The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: The N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expressions of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals. 42 refs., 5 figs.

  4. Early Loss of Telomerase Action in Yeast Creates a Dependence on the DNA Damage Response Adaptor Proteins

    PubMed Central

    Jay, Kyle A.; Smith, Dana L.

    2016-01-01

    Telomeres cap the ends of chromosomes, protecting them from degradation and inappropriate DNA repair processes that can lead to genomic instability. A short telomere elicits increased telomerase action on itself that replenishes telomere length, thereby stabilizing the telomere. In the prolonged absence of telomerase activity in dividing cells, telomeres eventually become critically short, inducing a permanent cell cycle arrest (senescence). We recently showed that even early after telomerase inactivation (ETI), yeast cells have accelerated mother cell aging and mildly perturbed cell cycles. Here, we show that the complete disruption of DNA damage response (DDR) adaptor proteins in ETI cells causes severe growth defects. This synthetic-lethality phenotype was as pronounced as that caused by extensive DNA damage in wild-type cells but showed genetic dependencies distinct from such damage and was completely alleviated by SML1 deletion, which increases deoxynucleoside triphosphate (dNTP) pools. Our results indicated that these deleterious effects in ETI cells cannot be accounted for solely by the slow erosion of telomeres due to incomplete replication that leads to senescence. We propose that normally occurring telomeric DNA replication stress is resolved by telomerase activity and the DDR in two parallel pathways and that deletion of Sml1 prevents this stress. PMID:27161319

  5. Early Loss of Telomerase Action in Yeast Creates a Dependence on the DNA Damage Response Adaptor Proteins.

    PubMed

    Jay, Kyle A; Smith, Dana L; Blackburn, Elizabeth H

    2016-07-15

    Telomeres cap the ends of chromosomes, protecting them from degradation and inappropriate DNA repair processes that can lead to genomic instability. A short telomere elicits increased telomerase action on itself that replenishes telomere length, thereby stabilizing the telomere. In the prolonged absence of telomerase activity in dividing cells, telomeres eventually become critically short, inducing a permanent cell cycle arrest (senescence). We recently showed that even early after telomerase inactivation (ETI), yeast cells have accelerated mother cell aging and mildly perturbed cell cycles. Here, we show that the complete disruption of DNA damage response (DDR) adaptor proteins in ETI cells causes severe growth defects. This synthetic-lethality phenotype was as pronounced as that caused by extensive DNA damage in wild-type cells but showed genetic dependencies distinct from such damage and was completely alleviated by SML1 deletion, which increases deoxynucleoside triphosphate (dNTP) pools. Our results indicated that these deleterious effects in ETI cells cannot be accounted for solely by the slow erosion of telomeres due to incomplete replication that leads to senescence. We propose that normally occurring telomeric DNA replication stress is resolved by telomerase activity and the DDR in two parallel pathways and that deletion of Sml1 prevents this stress. PMID:27161319

  6. The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants

    SciTech Connect

    Hipp, Katharina; Rau, Peter; Schäfer, Benjamin; Gronenborn, Bruno; Jeske, Holger

    2014-08-15

    Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clones prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast. - Highlights: • A potential cyclin interaction motif is conserved in geminivirus Rep proteins. • In ACMV Rep, this motif (RXL) is essential for rereplication of fission yeast DNA. • Mutating RXL abrogated viral infection completely in Nicotiana benthamiana. • Expression of a nanovirus Clink protein in yeast did not induce rereplication. • Plant viruses may have evolved multiple routes to exploit host DNA synthesis.

  7. Widespread compensatory evolution conserves DNA-encoded nucleosome organization in yeast.

    PubMed

    Kenigsberg, Ephraim; Bar, Amir; Segal, Eran; Tanay, Amos

    2010-01-01

    Evolution maintains organismal fitness by preserving genomic information. This is widely assumed to involve conservation of specific genomic loci among species. Many genomic encodings are now recognized to integrate small contributions from multiple genomic positions into quantitative dispersed codes, but the evolutionary dynamics of such codes are still poorly understood. Here we show that in yeast, sequences that quantitatively affect nucleosome occupancy evolve under compensatory dynamics that maintain heterogeneous levels of A+T content through spatially coupled A/T-losing and A/T-gaining substitutions. Evolutionary modeling combined with data on yeast polymorphisms supports the idea that these substitution dynamics are a consequence of weak selection. This shows that compensatory evolution, so far believed to affect specific groups of epistatically linked loci like paired RNA bases, is a widespread phenomenon in the yeast genome, affecting the majority of intergenic sequences in it. The model thus derived suggests that compensation is inevitable when evolution conserves quantitative and dispersed genomic functions. PMID:21203484

  8. Yeast RNA polymerase II initiates transcription in vitro at TATA sequences proximal to potential non-B forms of the DNA template.

    PubMed Central

    Lescure, B; Arcangioli, B

    1984-01-01

    Pure yeast RNA polymerase II selectively initiates an abortive in vitro transcript within a TATA box of the yeast iso-1 cytochrome c gene promoter. Using a series of promoter deletions we show that a DNA sequence located upstream of the TATA box is needed for an efficient in vitro transcription. Supercoiling of the DNA template is an absolute requirement for the specific in vitro transcription. Examination of the DNA structure near several in vitro initiation sites shows that the common features observed are the presence of a TATA sequence in which RNA synthesis is initiated, and which is proximal to a potential non-B form of the DNA (a B to Z transition or a cruciform structure). Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6376116

  9. The DNA-Binding Domain of Yeast Rap1 Interacts with Double-Stranded DNA in Multiple Binding Modes

    PubMed Central

    2015-01-01

    Saccharomyces cerevisiae repressor-activator protein 1 (Rap1) is an essential protein involved in multiple steps of DNA regulation, as an activator in transcription, as a repressor at silencer elements, and as a major component of the shelterin-like complex at telomeres. All the known functions of Rap1 require the known high-affinity and specific interaction of the DNA-binding domain with its recognition sequences. In this work, we focus on the interaction of the DNA-binding domain of Rap1 (Rap1DBD) with double-stranded DNA substrates. Unexpectedly, we found that while Rap1DBD forms a high-affinity 1:1 complex with its DNA recognition site, it can also form lower-affinity complexes with higher stoichiometries on DNA. These lower-affinity interactions are independent of the presence of the recognition sequence, and we propose they originate from the ability of Rap1DBD to bind to DNA in two different binding modes. In one high-affinity binding mode, Rap1DBD likely binds in the conformation observed in the available crystal structures. In the other alternative lower-affinity binding mode, we propose that a single Myb-like domain of the Rap1DBD makes interactions with DNA, allowing for more than one protein molecule to bind to the DNA substrates. Our findings suggest that the Rap1DBD does not simply target the protein to its recognition sequence but rather it might be a possible point of regulation. PMID:25382181

  10. Tel2p, a regulator of yeast telomeric length in vivo, binds to single-stranded telomeric DNA in vitro.

    PubMed

    Kota, R S; Runge, K W

    1999-09-01

    The telomeres of the yeast Saccharomyces cerevisiae consist of a duplex region of TG(1-3) repeats that acquire a single-stranded 3' extension of the TG(1-3) strand at the end of S-phase. The length of these repeats is kept within a defined range by regulators such as the TEL2-encoded protein (Tel2p). Here we show that Tel2p can specifically bind to single-stranded TG(1-3). Tel2p binding produced several shifted bands; however, only the slowest migrating band contained Tel2p. Methylation protection and interference experiments as well as gel shift experiments using inosine-containing probes indicated that the faster migrating bands resulted from Tel2p-mediated formation of DNA secondary structures held together by G-G interactions. Tel2p bound to single-stranded substrates that were at least 19 bases in length and contained 14 bases of TG(1-3), and also to double-stranded/single-stranded hybrid substrates with a 3' TG(1-3) overhang. Tel2p binding to a hybrid substrate with a 24 base single-stranded TG(1-3) extension also produced a band characteristic of G-G-mediated secondary structures. These data suggest that Tel2p could regulate telomeric length by binding to the 3' single-stranded TG(1-3) extension present at yeast telomeres. PMID:10525964

  11. Role of Inositol Phosphosphingolipid Phospholipase C1, the Yeast Homolog of Neutral Sphingomyelinases in DNA Damage Response and Diseases.

    PubMed

    Tripathi, Kaushlendra

    2015-01-01

    Sphingolipids play a very crucial role in many diseases and are well-known as signaling mediators in many pathways. Sphingolipids are produced during the de novo process in the ER (endoplasmic reticulum) from the nonsphingolipid precursor and comprise both structural and bioactive lipids. Ceramide is the central core of the sphingolipid pathway, and its production has been observed following various treatments that can induce several different cellular effects including growth arrest, DNA damage, apoptosis, differentiation, and senescence. Ceramides are generally produced through the sphingomyelin hydrolysis and catalyzed by the enzyme sphingomyelinase (SMase) in mammals. Presently, there are many known SMases and they are categorized into three groups acid SMases (aSMases), alkaline SMases (alk-SMASES), and neutral SMases (nSMases). The yeast homolog of mammalians neutral SMases is inositol phosphosphingolipid phospholipase C. Yeasts generally have inositol phosphosphingolipids instead of sphingomyelin, which may act as a homolog of mammalian sphingomyelin. In this review, we shall explain the structure and function of inositol phosphosphingolipid phospholipase C1, its localization inside the cells, mechanisms, and its roles in various cell responses during replication stresses and diseases. This review will also give a new basis for our understanding for the mechanisms and nature of the inositol phosphosphingolipid phospholipase C1/nSMase. PMID:26346287

  12. Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts.

    PubMed

    Adamczyk, Jagoda; Deregowska, Anna; Skoneczny, Marek; Skoneczna, Adrianna; Natkanska, Urszula; Kwiatkowska, Aleksandra; Rawska, Ewa; Potocki, Leszek; Kuna, Ewelina; Panek, Anita; Lewinska, Anna; Wnuk, Maciej

    2016-09-01

    The yeast strains of the Saccharomyces sensu stricto complex involved in beer production are a heterogeneous group whose genetic and genomic features are not adequately determined. Thus, the aim of the present study was to provide a genetic characterization of selected group of commercially available brewing yeasts both ale top-fermenting and lager bottom-fermenting strains. Molecular karyotyping revealed that the diversity of chromosome patterns and four strains with the most accented genetic variabilities were selected and subjected to genome-wide array-based comparative genomic hybridization (array-CGH) analysis. The differences in the gene copy number were found in five functional gene categories: (1) maltose metabolism and transport, (2) response to toxin, (3) siderophore transport, (4) cellular aldehyde metabolic process, and (5) L-iditol 2-dehydrogenase activity (p < 0.05). In the Saflager W-34/70 strain (Fermentis) with the most affected array-CGH profile, loss of aryl-alcohol dehydrogenase (AAD) gene dosage correlated with an imbalanced redox state, oxidative DNA damage and breaks, lower levels of nucleolar proteins Nop1 and Fob1, and diminished tolerance to fermentation-associated stress stimuli compared to other strains. We suggest that compromised stress response may not only promote oxidant-based changes in the nucleolus state that may affect fermentation performance but also provide novel directions for future strain improvement. PMID:27299603

  13. A 6. 5-Mb yeast artificial chromosome contig incorporating 33 DNA markers on the human X chromosome at Xq22

    SciTech Connect

    Vetrie, D.; Kendall, E.; Coffey, A.; Hassock, S.; Collins, J.; Todd, C.; Bobrow, M.; Bentley, D.R. ); Lehrach, H. ); Harris, A. )

    1994-01-01

    The Xq22 region of the human X chromosome contains genes for a number of inherited disorders. Sixty-nine yeast artificial chromosome clones have been isolated and assembled into a 6.5-Mb contig that contains 33 DNA markers localized to this region. This contig extends distally from DXS366 to beyond DXS87 and includes the genes involved in X-linked agammaglobulinemia (btk), Fabry disease (GLA), and Pelizaeus-Merzbacher disease (PLP). The order of markers in this contig is consistent with the known genetic and physical mapping information of Xq22. This cloned material provides a source from which to isolate other genes located in this part of the X chromosome. 45 refs., 2 figs., 2 tabs.

  14. Biochemical and genetic characterization of Hmi1p, a yeast DNA helicase involved in the maintenance of mitochondrial DNA.

    PubMed

    Monroe, Danny S; Leitzel, Adelaide K; Klein, Hannah L; Matson, Steven W

    2005-12-01

    The HMI1 gene encodes a DNA helicase that localizes to the mitochondria and is required for maintenance of the mitochondrial DNA (mtDNA) genome of Saccharomyces cerevisiae. Identified based on its homology with E. coli uvrD, the HMI1 gene product, Hmi1p, has been presumed to be involved in the replication of the 80 kb linear S. cerevisiae mtDNA genome. Here we report the purification of Hmi1p to apparent homogeneity and provide a characterization of the helicase reaction and the ATPase reaction with regard to NTP preference, divalent cation preference and the stimulatory effects of different nucleic acids on Hmi1p-catalysed ATPase activity. Genetic complementation assays indicate that mitochondrial localization of Hmi1p is essential for its role in mtDNA metabolism. The helicase activity, however, is not essential. Point mutants that lack ATPase/helicase activity partially complement a strain lacking Hmi1p. We suggest several possible roles for Hmi1p in mtDNA metabolism. PMID:16358299

  15. Dissection of the DNA binding domain of yeast Zn-finger protein Rme1p, a repressor of meiotic activator IME1.

    PubMed

    Shimizu, M; Hara, M; Murase, A; Shindo, H; Mitchell, A P

    1997-01-01

    A series of deletion mutants of the yeast Zn-finger protein Rme1p (Repressor of Meiosis) fused with maltose binding protein (MBP) were constructed, purified, and characterized to examine the DNA binding domain. It was shown by gel retardation assay that the DNA binding domain of Rme1p was attributed to C-terminal amino acid residues 171 to 300. All three Zn-fingers are involved in the DNA binding domain, but they are not sufficient for DNA binding ability. Notably, the C-terminal region (residues 285-300) is essential for DNA binding. Provided that the region folds into alpha-helix, the basic amino acid residues may form a ridge on one side of the helix, whereas the hydrophobic residues may form it on the other side. Thus, the DNA binding domain of Rme1p would be dissected two regions. The roles of C-terminal region in DNA recognition will be discussed. PMID:9586056

  16. Primary structure of yeast mitochondrial DNA-coded phenylalanine-tRNA.

    PubMed

    Martin, R P; Sibler, A P; Schneller, J M; Keith, G; Stahl, A J; Dirheimer, G

    1978-12-01

    Mitochondrial tRNAPhe from Saccharomyces cerevisiae isolated by two-dimensional gel electrophoresis was sequenced by fingerprinting uniformly labeled 32 P-tRNA as well as by 5'-end postlabeling techniques. Its sequence was found to be: pG-C-U-U-U-U-A-U-A-G-C-U-U-A-G-D-G-G-D-A-A-A-G-C-m22G-A-U-A-A-A-phi-U-G-A-A-m1G-A-phi-U-U-A-U-U-U-A-C-A-U-G-U-A-G-U-phi-C-G-A-U-U-C-U-C-A-U-U-A-A-G-G-G-C-A-C-C-A. The secondary structure we propose, in order to maximize base pairing in the phiC stem and to allow tertiary interaction between G15 and C46, excludes U50 from base pairing giving a bulge in the phiC stem. No conclusion can be drawn concerning the endosymbiotic theory of mitochondria evolution by comparing the primary structure of mt. tRNAPhe with other sequenced tRNAsPhe. This mt.tRNAPhe lacks some of the structural elements reported to be involved in the yeast cytoplasmic phenylalanyl-tRNA ligase recognition site and cannot be aminoacylated by purified yeast cytoplasmic phenylalanyl-tRNA ligase. PMID:370774

  17. Comparison of the Accuracy of Two Conventional Phenotypic Methods and Two MALDI-TOF MS Systems with That of DNA Sequencing Analysis for Correctly Identifying Clinically Encountered Yeasts

    PubMed Central

    Chao, Qiao-Ting; Lee, Tai-Fen; Teng, Shih-Hua; Peng, Li-Yun; Chen, Ping-Hung; Teng, Lee-Jene; Hsueh, Po-Ren

    2014-01-01

    We assessed the accuracy of species-level identification of two commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Bruker Biotyper and Vitek MS) and two conventional phenotypic methods (Phoenix 100 YBC and Vitek 2 Yeast ID) with that of rDNA gene sequencing analysis among 200 clinical isolates of commonly encountered yeasts. The correct identification rates of the 200 yeast isolates to species or complex (Candida parapsilosis complex, C. guilliermondii complex and C. rugosa complex) levels by the Bruker Biotyper, Vitek MS (using in vitro devices [IVD] database), Phoenix 100 YBC and Vitek 2 Yeast ID (Sabouraud's dextrose agar) systems were 92.5%, 79.5%, 89%, and 74%, respectively. An additional 72 isolates of C. parapsilosis complex and 18 from the above 200 isolates (30 in each of C. parapsilosis, C. metapsilosis, and C. orthopsilosis) were also evaluated separately. Bruker Biotyper system could accurately identify all C. parapsilosis complex to species level. Using Vitek 2 MS (IVD) system, all C. parapsilosis but none of C. metapsilosis, or C. orthopsilosis could be accurately identified. Among the 89 yeasts misidentified by the Vitek 2 MS (IVD) system, 39 (43.8%), including 27 C. orthopsilosis isolates, could be correctly identified Using the Vitek MS Plus SARAMIS database for research use only. This resulted in an increase in the rate of correct identification of all yeast isolates (87.5%) by Vitek 2 MS. The two species in C. guilliermondii complex (C. guilliermondii and C. fermentati) isolates were correctly identified by cluster analysis of spectra generated by the Bruker Biotyper system. Based on the results obtained in the current study, MALDI-TOF MS systems present a promising alternative for the routine identification of yeast species, including clinically commonly and rarely encountered yeast species and several species belonging to C. parapsilosis complex, C. guilliermondii complex

  18. Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain.

    PubMed Central

    Kirkpatrick, D T; Fan, Q; Petes, T D

    1999-01-01

    The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot. Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination. We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of recombination hotspots, alpha (transcription factor dependent) and beta (transcription factor independent). PMID:10224246

  19. Identification of Novel Protein–Ligand Interactions by Exon Microarray Analysis of Yeast Surface Displayed cDNA Library Selection Outputs

    PubMed Central

    Bidlingmaier, Scott; Liu, Bin

    2016-01-01

    Yeast surface display is widely utilized to screen large libraries for proteins or protein fragments with specific binding properties. We have previously constructed and utilized yeast surface displayed human cDNA libraries to identify protein fragments that bind to various target ligands. Conventional approaches employ monoclonal screening and sequencing of polyclonal outputs that have been enriched for binding to a target molecule by several rounds of affinity-based selection. Frequently, a small number of clones will dominate the selection output, making it difficult to comprehensively identify potentially important interactions due to low representation in the selection output. We have developed a novel method to address this problem. By analyzing selection outputs using high-density human exon microarrays, the full potential of selection output diversity can be revealed in one experiment. FACS-based selection using yeast surface displayed human cDNA libraries combined with exon microarray analysis of the selection outputs is a powerful way of rapidly identifying protein fragments with affinity for any soluble ligand that can be fluorescently detected, including small biological molecules and drugs. In this report we present protocols for exon microarray-based analysis of yeast surface display human cDNA library selection outputs. PMID:26060075

  20. DNA repair defects sensitize cells to anticodon nuclease yeast killer toxins.

    PubMed

    Klassen, Roland; Wemhoff, Sabrina; Krause, Jens; Meinhardt, Friedhelm

    2011-03-01

    Killer toxins from Kluyveromyces lactis (zymocin) and Pichia acaciae (PaT) were found to disable translation in target cells by virtue of anticodon nuclease (ACNase) activities on tRNA(Glu) and tRNA(Gln), respectively. Surprisingly, however, ACNase exposure does not only impair translation, but also affects genome integrity and concomitantly DNA damage occurs. Previously, it was shown that homologous recombination protects cells from ACNase toxicity. Here, we have analyzed whether other DNA repair pathways are functional in conferring ACNase resistance as well. In addition to HR, base excision repair (BER) and postreplication repair (PRR) promote clear resistance to either, PaT and zymocin. Comparative toxin sensitivity analysis of BER mutants revealed that its ACNase protective function is due to the endonucleases acting on apurinic (AP) sites, whereas none of the known DNA glycosylases is involved. Because PaT and zymocin require the presence of the ELP3/TRM9-dependent wobble uridine modification 5-methoxy-carbonyl-methyl (mcm(5)) for tRNA cleavage, we analyzed toxin response in DNA repair mutants additionally lacking such tRNA modifications. ACNase resistance caused by elp3 or trm9 mutations was found to rescue hypersensitivity of DNA repair defects, consistent with DNA damage to occur as a consequence of tRNA cleavage. The obtained genetic evidence promises to reveal new aspects into the mechanism linking translational fidelity and genome surveillance. PMID:21188417

  1. Cernunnos interacts with the XRCC4 x DNA-ligase IV complex and is homologous to the yeast nonhomologous end-joining factor Nej1.

    PubMed

    Callebaut, Isabelle; Malivert, Laurent; Fischer, Alain; Mornon, Jean-Paul; Revy, Patrick; de Villartay, Jean-Pierre

    2006-05-19

    DNA double strand breaks are considered as the most harmful DNA lesions and are repaired by either homologous recombination or nonhomologous end joining (NHEJ). A new NHEJ factor, Cernunnos, has been identified, the defect of which leads to a severe immunodeficiency condition associated with microcephaly and other developmental defects in humans. This presentation is reminiscent to that of DNA-ligase IV deficiency and suggests a possible interplay between Cernunnos and the XRCC4 x DNA-ligase IV complex. We show here that Cernunnos physically interacts with the XRCC4 x DNA-ligase IV complex. Moreover, a combination of sensitive methods of sequence analysis revealed that Cernunnos can be associated with the XRCC4 family of proteins and that it corresponds to the genuine homolog of the yeast Nej1 protein. Altogether these results shed new lights on the last step, the DNA religation, of the NHEJ pathway. PMID:16571728

  2. A new set of rDNA-NTS-based multiple integrative cassettes for the development of antibiotic-marker-free recombinant yeasts.

    PubMed

    Moon, Hye Yun; Lee, Dong Wook; Sim, Gyu Hun; Kim, Hong-Jin; Hwang, Jee Youn; Kwon, Mun-Gyeong; Kang, Bo-Kyu; Kim, Jong Man; Kang, Hyun Ah

    2016-09-10

    The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5' and 3'-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to ∼30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring ∼30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption. PMID:27411901

  3. Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA

    PubMed Central

    Nakamura, Takahiro; Pluskal, Tomáš; Nakaseko, Yukinobu; Yanagida, Mitsuhiro

    2012-01-01

    Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA. PMID:23091701

  4. A role for DNA polymerase α in epigenetic control of transcriptional silencing in fission yeast

    PubMed Central

    Nakayama, Jun-ichi; Allshire, Robin C.; Klar, Amar J.S.; Grewal, Shiv I.S.

    2001-01-01

    In the fission yeast Schizosaccharomyces pombe, transcriptional silencing at the mating-type region, centromeres and telomeres is epigenetically controlled, and results from the assembly of higher order chromatin structures. Chromatin proteins associated with these silenced loci are believed to serve as molecular bookmarks that help promote inheritance of the silenced state during cell division. Specifically, a chromodomain protein Swi6 is believed to be an important determinant of the epigenetic imprint. Here, we show that a mutation in DNA polymerase α (polα) affects Swi6 localization at the mating-type region and causes a 45-fold increase in spontaneous transition from the silenced epigenetic state to the expressed state. We also demonstrate that polα mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polα and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Polα in vitro. Moreover, silencing-defective mutant Polα displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Polα, in heterochromatin assembly and inheritance of epigenetic chromatin structures. PMID:11387218

  5. Condensin HEAT Subunits Required for DNA Repair, Kinetochore/Centromere Function and Ploidy Maintenance in Fission Yeast

    PubMed Central

    Xu, Xingya; Nakazawa, Norihiko; Yanagida, Mitsuhiro

    2015-01-01

    Condensin, a central player in eukaryotic chromosomal dynamics, contains five evolutionarily-conserved subunits. Two SMC (structural maintenance of chromosomes) subunits contain ATPase, hinge, and coiled-coil domains. One non-SMC subunit is similar to bacterial kleisin, and two other non-SMC subunits contain HEAT (similar to armadillo) repeats. Here we report isolation and characterization of 21 fission yeast (Schizosaccharomyces pombe) mutants for three non-SMC subunits, created using error-prone mutagenesis that resulted in single-amino acid substitutions. Beside condensation, segregation, and DNA repair defects, similar to those observed in previously isolated SMC and cnd2 mutants, novel phenotypes were observed for mutants of HEAT-repeats containing Cnd1 and Cnd3 subunits. cnd3-L269P is hypersensitive to the microtubule poison, thiabendazole, revealing defects in kinetochore/centromere and spindle assembly checkpoints. Three cnd1 and three cnd3 mutants increased cell size and doubled DNA content, thereby eliminating the haploid state. Five of these mutations reside in helix B of HEAT repeats. Two non-SMC condensin subunits, Cnd1 and Cnd3, are thus implicated in ploidy maintenance. PMID:25764183

  6. [Expression Of DNA-Encoded Antidote to Organophosphorus Toxins in the Methylotrophic Yeast Pichia Pastoris].

    PubMed

    Terekhov, S S; Bobik, T V; Mokrushina, Yu A; Stepanova, A V; Aleksandrova, N M; Smirnov, I V; Belogurov, A A; Ponomarenko, N A; Gabibov, A G

    2016-01-01

    A platform for the cloning and expression of active human butyrylcholinesterase (BuChE) in the yeast Pichia pastoris is first presented. Genetic constructs for BuChE gene expression, separately and in conjunction with a proline-rich peptide called proline-rich attachment domain (PRAD), are based on the vector pPICZαA. It is shown that the highest level of production is achieved in the expression of a BuChE gene without PRAD pPICZαA. It is found that one can obtain up to 125 mg of active enzyme from 1 L of culture medium at an optimal pH environment (pH 7.6), an optical seed culture density of 3 o.u., and an optimum methanol addition mode of (0.5% methanol in the first day and 0.2% thereafter from the second day). PMID:27266247

  7. Yeast linker histone Hho1p is required for efficient RNA polymerase I processivity and transcriptional silencing at the ribosomal DNA

    PubMed Central

    Levy, Anat; Eyal, Miri; Hershkovits, Gitit; Salmon-Divon, Mali; Klutstein, Michael; Katcoff, Don Jay

    2008-01-01

    Nucleosome core particles in eukaryotes are linked by a stretch of DNA that is usually associated with a linker histone. Here, we show in yeast, that the presence of yeast linker histone Hho1p represses expression of a pol II transcribed gene (MET15) embedded in the rDNA. In vivo deletions of Hho1p sequences showed that the second globular domain is sufficient for that repression, whereas the presence of the N terminus is required for its derepression. In contrast, a run-on assay confirmed by a ChIP experiment showed that Hho1p is required for maximal pol I processivity during rDNA transcription. Psoralen accessibility experiments indicated that Hho1p is necessary for normal rDNA compaction. DNA array expression analysis comparing RNA transcripts in wild-type and hho1 strains before and after a heat-shock showed that Hho1p is necessary to achieve wild-type mRNA levels of transcripts that encode ribosomal components. Taken together, our results suggest that Hho1p is involved in rDNA compaction, and like core histones, is required for efficient rDNA transcription by pol I. PMID:18687885

  8. Characterization of vanadate-dependent NADH oxidation activity and isolation of yeast DNA which complements a class 1 vanadate resistance mutation

    SciTech Connect

    Minasi, L.E.

    1989-01-01

    A vanadate-dependent NADH oxidation activity has been characterized in plasma membranes from the yeast S cerevisiae. NADH oxidation activity was maximally stimulated at pH 5.0 in phosphate buffer. NADH oxidation was not dependent on the concentration of plasma membranes. The vanadate-dependent NADH oxidation activity was abolished under anaerobic conditions and the concomitant uptake of oxygen occurred during NADH oxidation. The activity was inhibited by superoxide dismutase and stimulated by the presence of paraquat. These results indicate that the vanadate stimulation of NADH oxidation in yeast plasma membranes occurs as a result of the vanadate-dependent oxidation of NADH by superoxide, generated by a plasma membrane NADH oxidase. {sup 51}V-NMR results indicated that a phosphate-vanadate anhydride was the stimulatory species in pH 5.0 and pH 7.0 phosphate buffer. Yeast DNA has been isolated which complements a class 1 vanadate resistance mutation.

  9. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation

    PubMed Central

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J.; Fox, Catherine A.

    2016-01-01

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  10. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

    PubMed

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J; Fox, Catherine A

    2016-01-01

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  11. DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain.

    PubMed

    Morrill, Summer A; Exner, Alexandra E; Babokhov, Michael; Reinfeld, Bradley I; Fuchs, Stephen M

    2016-05-27

    The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24-26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1 In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length. PMID:27026700

  12. DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain*

    PubMed Central

    Morrill, Summer A.; Exner, Alexandra E.; Babokhov, Michael; Reinfeld, Bradley I.

    2016-01-01

    The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae. First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24–26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1. In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length. PMID:27026700

  13. Sen1, the yeast homolog of human senataxin, plays a more direct role than Rad26 in transcription coupled DNA repair.

    PubMed

    Li, Wentao; Selvam, Kathiresan; Rahman, Sheikh A; Li, Shisheng

    2016-08-19

    Rad26, a DNA dependent ATPase that is homologous to human CSB, has been well known to play an important role in transcription coupled DNA repair (TCR) in the yeast Saccharomyces cerevisiae Sen1, a DNA/RNA helicase that is essential for yeast cell viability and homologous to human senataxin, has been known to be required for transcriptional termination of short noncoding RNA genes and for a fail-safe transcriptional termination mechanism of protein-coding genes. Sen1 has also been shown to protect the yeast genome from transcription-associated recombination by resolving RNA:DNA hybrids naturally formed during transcription. Here, we show that the N-terminal non-essential region of Sen1 plays an important role in TCR, whereas the C-terminal nonessential region and the helicase activity of Sen1 are largely dispensable for the repair. Unlike Rad26, which becomes completely dispensable for TCR in cells lacking the TCR repressor Spt4, Sen1 is still required for efficient TCR in the absence of Spt4. Also unlike Rad26, which is important for repair at many but not all damaged sites in the transcribed strand of a gene, Sen1 is required for efficient repair at essentially all the damaged sites. Our results indicate that Sen1 plays a more direct role than Rad26 in TCR. PMID:27179024

  14. In situ hybridization to cytogenetic bands of yeast artificial chromosomes covering 50% of human Xq24-Xq28 DNA

    PubMed Central

    Montanaro, Vittorio; Casamassimi, Amelia; D'Urso, Michele; Yoon, Jae-Young; Freije, Wadiha; Schlessinger, David; Muenke, Maximilian; Nussbaum, Robert L.; Saccone, Salvatore; Maugeri, Silvana; Santoro, Anna Maria; Motta, Salvatore; Della Valle, Giuliano

    1991-01-01

    From the collection described by Abidi et al., 102 yeast artificial chromosomes (YACs) with human DNA inserts more than 300 kb in length were assigned to chromosomal band positions on early metaphase chromosomes by in situ hybridization using the biotin-avidin method. All the YACs hybridized within the Xq24-Xqter region, supporting the origin of the vast majority of the YACs from single human X-chromosomal sites. With assignments precise to ±0.5 bands, YACs were distributed among cytogenetic bands to roughly equal extents. Thus, there is no gross bias in the cloning of DNA from different bands into large YACs. To test band assignments further, hybridizations were carried out blind, and band positions were then compared with (1) probe localizations in cases in which a reported location was present in one of the YACs; (2) cross-hybridization of a labeled YAC with others in the collection; and (3) hybridization to a panel of DNAs from a series of hybrid cells containing Xq DNA truncated at various regions. Of 31 cases in which YACs contained a probe with a previously reported location, 28 in situ assignments were in agreement, and 14 other assignments, including one of the three discordant with probe localization, were confirmed by YAC cross-hybridization studies. Results with a group of nine YACs were further confirmed with a panel of somatic cell hybrid DNAs from that region. Five YACs hybridized both to Xq25 and to a second site (four in Xq27 and one in Xq28), suggestive of some duplication of DNA of the hybrid cell and perhaps in normal X chromosomes. The in situ assignments are thus sufficient to place YACs easily and systematically within bins of about 7–10 Mb and to detect some possible anomalies. Furthermore, on the basis of expectations for random cloning of DNA in YACs, the assigned YACs probably cover more than 50% of the total Xq24-Xq28 region. This provides one way to initiate the assembly of YAC contigs over extended chromosomal regions. Images

  15. Rapid Identification of Chemoresistance Mechanisms Using Yeast DNA Mismatch Repair Mutants.

    PubMed

    Ojini, Irene; Gammie, Alison

    2015-09-01

    Resistance to cancer therapy is a major obstacle in the long-term treatment of cancer. A greater understanding of drug resistance mechanisms will ultimately lead to the development of effective therapeutic strategies to prevent resistance from occurring. Here, we exploit the mutator phenotype of mismatch repair defective yeast cells combined with whole genome sequencing to identify drug resistance mutations in key pathways involved in the development of chemoresistance. The utility of this approach was demonstrated via the identification of the known CAN1 and TOP1 resistance targets for two compounds, canavanine and camptothecin, respectively. We have also experimentally validated the plasma membrane transporter HNM1 as the primary drug resistance target of mechlorethamine. Furthermore, the sequencing of mitoxantrone-resistant strains identified inactivating mutations within IPT1, a gene encoding inositolphosphotransferase, an enzyme involved in sphingolipid biosynthesis. In the case of bactobolin, a promising anticancer drug, the endocytosis pathway was identified as the drug resistance target responsible for conferring resistance. Finally, we show that that rapamycin, an mTOR inhibitor previously shown to alter the fitness of the ipt1 mutant, can effectively prevent the formation of mitoxantrone resistance. The rapid and robust nature of these techniques, using Saccharomyces cerevisiae as a model organism, should accelerate the identification of drug resistance targets and guide the development of novel therapeutic combination strategies to prevent the development of chemoresistance in various cancers. PMID:26199284

  16. Deficiencies in mitochondrial DNA compromise the survival of yeast cells at critically high temperatures.

    PubMed

    Zubko, Elena I; Zubko, Mikhajlo K

    2014-01-01

    To address possible roles of mitochondrial genes in adaptation of eukaryotic cells to critical temperatures, we compared thermotolerance of mitochondrial rho mutants and wild type cells of six rho positive yeast species: Candida glabrata, Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces paradoxus and Saccharomyces pastorianus. All rho mutants manifested compromised thermotolerance as a common phenotype. Analysis of viabilities at critical temperatures (32-45 °C) showed the reduction of maximum permissive temperatures (MPTs) in all rho mutants in comparison to their wild type counterparts. Degrees of the compromised thermotolerance depended on maximum permissive temperatures for wild type cells: the highest levels of MPT reductions for rho mutants took place in species and strains with highest MPTs for wild types. Short term exposures of S. cerevisiae cells (up to 3.5h) at non-permissive temperatures (45 °C and 50 °C) also lead to more rapid cell death of rho mutants as compared to wild type cells. We conclude that: (1) compromised thermotolerance could be a generic phenotypic property of rho mutants; (2) the enhanced thermotolerance of cells possessing mitochondrial genomes could be one of selective advantages in adaptation to environmental factors, in particular to enhanced temperatures. PMID:23890722

  17. Rapid Identification of Chemoresistance Mechanisms Using Yeast DNA Mismatch Repair Mutants

    PubMed Central

    Ojini, Irene; Gammie, Alison

    2015-01-01

    Resistance to cancer therapy is a major obstacle in the long-term treatment of cancer. A greater understanding of drug resistance mechanisms will ultimately lead to the development of effective therapeutic strategies to prevent resistance from occurring. Here, we exploit the mutator phenotype of mismatch repair defective yeast cells combined with whole genome sequencing to identify drug resistance mutations in key pathways involved in the development of chemoresistance. The utility of this approach was demonstrated via the identification of the known CAN1 and TOP1 resistance targets for two compounds, canavanine and camptothecin, respectively. We have also experimentally validated the plasma membrane transporter HNM1 as the primary drug resistance target of mechlorethamine. Furthermore, the sequencing of mitoxantrone-resistant strains identified inactivating mutations within IPT1, a gene encoding inositolphosphotransferase, an enzyme involved in sphingolipid biosynthesis. In the case of bactobolin, a promising anticancer drug, the endocytosis pathway was identified as the drug resistance target responsible for conferring resistance. Finally, we show that that rapamycin, an mTOR inhibitor previously shown to alter the fitness of the ipt1 mutant, can effectively prevent the formation of mitoxantrone resistance. The rapid and robust nature of these techniques, using Saccharomyces cerevisiae as a model organism, should accelerate the identification of drug resistance targets and guide the development of novel therapeutic combination strategies to prevent the development of chemoresistance in various cancers. PMID:26199284

  18. Involvement of budding yeast Rad5 in translesion DNA synthesis through physical interaction with Rev1

    PubMed Central

    Xu, Xin; Lin, Aiyang; Zhou, Cuiyan; Blackwell, Susan R.; Zhang, Yiran; Wang, Zihao; Feng, Qianqian; Guan, Ruifang; Hanna, Michelle D.; Chen, Zhucheng; Xiao, Wei

    2016-01-01

    DNA damage tolerance (DDT) is responsible for genomic stability and cell viability by bypassing the replication block. In Saccharomyces cerevisiae DDT employs two parallel branch pathways to bypass the DNA lesion, namely translesion DNA synthesis (TLS) and error-free lesion bypass, which are mediated by sequential modifications of PCNA. Rad5 has been placed in the error-free branch of DDT because it contains an E3 ligase domain required for PCNA polyubiquitination. Rad5 is a multi-functional protein and may also play a role in TLS, since it interacts with the TLS polymerase Rev1. In this study we mapped the Rev1-interaction domain in Rad5 to the amino acid resolution and demonstrated that Rad5 is indeed involved in TLS possibly through recruitment of Rev1. Genetic analyses show that the dual functions of Rad5 can be separated and reconstituted. Crystal structure analysis of the Rad5–Rev1 interaction reveals a consensus RFF motif in the Rad5 N-terminus that binds to a hydrophobic pocket within the C-terminal domain of Rev1 that is highly conserved in eukaryotes. This study indicates that Rad5 plays a critical role in pathway choice between TLS and error-free DDT. PMID:27001510

  19. Dual requirement for the yeast MMS19 gene in DNA repair and RNA polymerase II transcription.

    PubMed Central

    Lauder, S; Bankmann, M; Guzder, S N; Sung, P; Prakash, L; Prakash, S

    1996-01-01

    Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the involvement of a large number of protein factors in nucleotide excision repair (NER) of UV-damaged DNA. However, how MMS19 affects this process has remained unclear. Here, we report on the isolation of the MMS19 gene and the determination of its role in NER and other cellular processes. Genetic and biochemical evidence indicates that besides its function in NER, MMS19 also affects RNA polymerase II (Pol II) transcription. mms19delta cells do not grow at 37 degrees C, and mutant extract exhibits a thermolabile defect in Pol II transcription. Thus, Mms19 protein resembles TFIIH in that it is required for both transcription and DNA repair. However, addition of purified Mms19 protein does not alleviate the transcriptional defect of the mms19delta extract, nor does it stimulate the incision of UV-damaged DNA reconstituted from purified proteins. Interestingly, addition of purified TFIIH corrects the transcriptional defect of the mms19delta extract. Mms19 is, however, not a component of TFIIH or of Pol II holoenzyme. These and other results suggest that Mms19 affects NER and transcription by influencing the activity of TFIIH as an upstream regulatory element. It is proposed that mutations in the human MMS19 counterpart could result in syndromes in which both NER and transcription are affected. PMID:8943333

  20. Involvement of budding yeast Rad5 in translesion DNA synthesis through physical interaction with Rev1.

    PubMed

    Xu, Xin; Lin, Aiyang; Zhou, Cuiyan; Blackwell, Susan R; Zhang, Yiran; Wang, Zihao; Feng, Qianqian; Guan, Ruifang; Hanna, Michelle D; Chen, Zhucheng; Xiao, Wei

    2016-06-20

    DNA damage tolerance (DDT) is responsible for genomic stability and cell viability by bypassing the replication block. In Saccharomyces cerevisiae DDT employs two parallel branch pathways to bypass the DNA lesion, namely translesion DNA synthesis (TLS) and error-free lesion bypass, which are mediated by sequential modifications of PCNA. Rad5 has been placed in the error-free branch of DDT because it contains an E3 ligase domain required for PCNA polyubiquitination. Rad5 is a multi-functional protein and may also play a role in TLS, since it interacts with the TLS polymerase Rev1. In this study we mapped the Rev1-interaction domain in Rad5 to the amino acid resolution and demonstrated that Rad5 is indeed involved in TLS possibly through recruitment of Rev1. Genetic analyses show that the dual functions of Rad5 can be separated and reconstituted. Crystal structure analysis of the Rad5-Rev1 interaction reveals a consensus RFF motif in the Rad5 N-terminus that binds to a hydrophobic pocket within the C-terminal domain of Rev1 that is highly conserved in eukaryotes. This study indicates that Rad5 plays a critical role in pathway choice between TLS and error-free DDT. PMID:27001510

  1. DNA topoisomerase III localizes to centromeres and affects centromeric CENP-A levels in fission yeast.

    PubMed

    Norman-Axelsson, Ulrika; Durand-Dubief, Mickaël; Prasad, Punit; Ekwall, Karl

    2013-01-01

    Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology, and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-A(Cnp1) at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays, we show that Top3, unlike Top1 and Top2, is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-A(Cnp1) occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-A(Cnp1) at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-A(Cnp1) in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology, which in turn affects the dynamics of CENP-A(Cnp1) nucleosomes. PMID:23516381

  2. DNA Topoisomerase III Localizes to Centromeres and Affects Centromeric CENP-A Levels in Fission Yeast

    PubMed Central

    Norman-Axelsson, Ulrika; Durand-Dubief, Mickaël; Prasad, Punit; Ekwall, Karl

    2013-01-01

    Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology, and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-ACnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays, we show that Top3, unlike Top1 and Top2, is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-ACnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-ACnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-ACnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology, which in turn affects the dynamics of CENP-ACnp1 nucleosomes. PMID:23516381

  3. Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

    PubMed Central

    Li, Ping; Jin, Hui; Yu, Hong-Guo

    2014-01-01

    During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240

  4. NMR chemical shift perturbation mapping of DNA binding by a zinc-finger domain from the yeast transcription factor ADR1.

    PubMed Central

    Schmiedeskamp, M.; Rajagopal, P.; Klevit, R. E.

    1997-01-01

    Mutagenesis studies have revealed that the minimal DNA-binding domain of the yeast transcription factor ADR1 consists of two Cys2-His2 zinc fingers plus an additional 20 residues proximal and N-terminal to the fingers. We have assigned NMR 1H, 15N, and 13C chemical shifts for the entire minimal DNA-binding domain of ADR1 both free and bound to specific DNA. 1H chemical shift values suggest little structural difference between the zinc fingers in this construct and in single-finger constructs, and 13C alpha chemical shift index analysis indicates little change in finger structure upon DNA binding. 1H chemical shift perturbations upon DNA binding are observed, however, and these are mapped to define the protein-DNA interface. The two zinc fingers appear to bind DNA with different orientations, as the entire helix of finger 1 is perturbed, while only the extreme N-terminus of the finger 2 helix is affected. Furthermore, residues N-terminal to the first finger undergo large chemical shift changes upon DNA binding suggesting a role at the protein-DNA interface. A striking correspondence is observed between the protein-DNA interface mapped by chemical shift changes and that previously mapped by mutagenesis. PMID:9300483

  5. Structural analysis of the interactions between hsp70 chaperones and the yeast DNA replication protein Orc4p.

    PubMed

    Moreno-del Alamo, María; Sánchez-Gorostiaga, Alicia; Serrano, Ana M; Prieto, Alicia; Cuéllar, Jorge; Martín-Benito, Jaime; Valpuesta, José M; Giraldo, Rafael

    2010-10-15

    Hsp70 chaperones, besides their role in assisting protein folding, are key modulators of protein disaggregation, being consistently found as components of most macromolecular assemblies isolated in proteome-wide affinity purifications. A wealth of structural information has been recently acquired on Hsp70s complexed with Hsp40 and NEF co-factors and with small hydrophobic target peptides. However, knowledge of how Hsp70s recognize large protein substrates is still limited. Earlier, we reported that homologue Hsp70 chaperones (DnaK in Escherichia coli and Ssa1-4p/Ssb1-2p in Saccharomyces cerevisiae) bind strongly, both in vitro and in vivo, to the AAA+ domain in the Orc4p subunit of yeast origin recognition complex (ORC). ScORC is the paradigm for eukaryotic DNA replication initiators and consists of six distinct protein subunits (ScOrc1p-ScOrc 6p). Here, we report that a hydrophobic sequence (IL(4)) in the initiator specific motif (ISM) in Orc4p is the main target for DnaK/Hsp70. The three-dimensional electron microscopy reconstruction of a stable Orc4p(2)-DnaK complex suggests that the C-terminal substrate-binding domain in the chaperone clamps the AAA+ IL(4) motif in one Orc4p molecule, with the substrate-binding domain lid subdomain wedging apart the other Orc4p subunit. Pairwise co-expression in E. coli shows that Orc4p interacts with Orc1/2/5p. Mutation of IL(4) selectively disrupts Orc4p interaction with Orc2p. Allelic substitution of ORC4 by mutants in each residue of IL(4) results in lethal (I184A) or thermosensitive (L185A and L186A) initiation-defective phenotypes in vivo. The interplay between Hsp70 chaperones and the Orc4p-IL(4) motif might have an adaptor role in the sequential, stoichiometric assembly of ScORC subunits. PMID:20732327

  6. Genomic heterogeneity and structural variation in soybean near isogenic lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Near-isogenic lines (NILs) are a critical genetic resource for the soybean research community. The ability to identify and characterize the genes driving the phenotypic differences between NILs is limited by the degree to which differential genetic introgressions can be resolved. Furthermore, the ge...

  7. A yeast-endonuclease-generated DNA break induces antigenic switching in Trypanosoma brucei.

    PubMed

    Boothroyd, Catharine E; Dreesen, Oliver; Leonova, Tatyana; Ly, K Ina; Figueiredo, Luisa M; Cross, George A M; Papavasiliou, F Nina

    2009-05-14

    Trypanosoma brucei is the causative agent of African sleeping sickness in humans and one of the causes of nagana in cattle. This protozoan parasite evades the host immune system by antigenic variation, a periodic switching of its variant surface glycoprotein (VSG) coat. VSG switching is spontaneous and occurs at a rate of about 10(-2)-10(-3) per population doubling in recent isolates from nature, but at a markedly reduced rate (10(-5)-10(-6)) in laboratory-adapted strains. VSG switching is thought to occur predominantly through gene conversion, a form of homologous recombination initiated by a DNA lesion that is used by other pathogens (for example, Candida albicans, Borrelia sp. and Neisseria gonorrhoeae) to generate surface protein diversity, and by B lymphocytes of the vertebrate immune system to generate antibody diversity. Very little is known about the molecular mechanism of VSG switching in T. brucei. Here we demonstrate that the introduction of a DNA double-stranded break (DSB) adjacent to the approximately 70-base-pair (bp) repeats upstream of the transcribed VSG gene increases switching in vitro approximately 250-fold, producing switched clones with a frequency and features similar to those generated early in an infection. We were also able to detect spontaneous DSBs within the 70-bp repeats upstream of the actively transcribed VSG gene, indicating that a DSB is a natural intermediate of VSG gene conversion and that VSG switching is the result of the resolution of this DSB by break-induced replication. PMID:19369939

  8. Telomere-related functions of yeast KU in the repair of bleomycin-induced DNA damage.

    PubMed

    Tam, Angela T Y; Pike, Brietta L; Hammet, Andrew; Heierhorst, Jörg

    2007-06-01

    Bleomycins are small glycopeptide cancer chemotherapeutics that give rise to 3'-modified DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, DSBs are predominantly repaired by RAD52-dependent homologous recombination (HR) with some support by Yku70/Yku80 (KU)-dependent pathways. The main DSB repair function of KU is believed to be as part of the non-homologous end-joining (NHEJ) pathway, but KU also functions in a "chromosome healing" pathway that seals DSBs by de novo telomere addition. We report here that rad52Deltayku70Delta double mutants are considerably more bleomycin hypersensitive than rad52Deltalig4Delta cells that lack the NHEJ-specific DNA ligase 4. Moreover, the telomere-specific KU mutation yku80-135i also dramatically increases rad52Delta bleomycin hypersensitivity, almost to the level of rad52Deltayku80Delta. The results indicate that telomere-specific functions of KU play a more prominent role in the repair of bleomycin-induced damage than its NHEJ functions, which could have important clinical implications for bleomycin-based combination chemotherapies. PMID:17442269

  9. Comparative Analysis of in vivo Interactions Between Rev1 Protein and Other Y-Family DNA Polymerases in Animals and Yeasts

    PubMed Central

    Kosarek, J. Nicole; Woodruff, Rachel V.; Rivera-Begeman, Amanda; Guo, Caixia; D’Souza, Sanjay; Koonin, Eugene V.; Walker, Graham C.; Friedberg, Errol C.

    2008-01-01

    Summary Eukaryotes are endowed with multiple specialized DNA polymerases, some (if not all) of which are believed to play important roles in the tolerance of base damage during DNA replication. Among these DNA polymerases, Rev1 protein (a deoxycytidyl transferase) from vertebrates interacts with several other specialized polymerases via a highly conserved C-terminal region. The present studies assessed whether these interactions are retained in more experimentally tractable model systems, including yeasts, flies, and the nematode C. elegans. We observed a physical interaction between Rev1 protein and other Y-family polymerases in the fruit fly Drosophila melanogaster. However, despite the fact that the C-terminal region of Drosophila and yeast Rev1 are conserved from vertebrates to a similar extent, such interactions were not observed in S. cerevisiae or S. pombe. With respect to regions in specialized DNA polymerases that are required for interaction with Rev1, we find predicted disorder to be an underlying structural commonality. The results of this study suggest that special consideration should be exercised when making mechanistic extrapolations regarding translesion DNA synthesis from one eukaryotic system to another. PMID:18242152

  10. Sequencer-Based Capillary Gel Electrophoresis (SCGE) Targeting the rDNA Internal Transcribed Spacer (ITS) Regions for Accurate Identification of Clinically Important Yeast Species

    PubMed Central

    Chen, Sharon C.-A.; Wang, He; Zhang, Li; Fan, Xin; Xu, Zhi-Peng; Cheng, Jing-Wei; Kong, Fanrong; Zhao, Yu-Pei; Xu, Ying-Chun

    2016-01-01

    Accurate species identification of Candida, Cryptococcus, Trichosporon and other yeast pathogens is important for clinical management. In the present study, we developed and evaluated a yeast species identification scheme by determining the rDNA internal transcribed spacer (ITS) region length types (LTs) using a sequencer-based capillary gel electrophoresis (SCGE) approach. A total of 156 yeast isolates encompassing 32 species were first used to establish a reference SCGE ITS LT database. Evaluation of the ITS LT database was then performed on (i) a separate set of (n = 97) clinical isolates by SCGE, and (ii) 41 isolates of 41 additional yeast species from GenBank by in silico analysis. Of 156 isolates used to build the reference database, 41 ITS LTs were identified, which correctly identified 29 of the 32 (90.6%) species, with the exception of Trichosporon asahii, Trichosporon japonicum and Trichosporon asteroides. In addition, eight of the 32 species revealed different electropherograms and were subtyped into 2–3 different ITS LTs each. Of the 97 test isolates used to evaluate the ITS LT scheme, 96 (99.0%) were correctly identified to species level, with the remaining isolate having a novel ITS LT. Of the additional 41 isolates for in silico analysis, none was misidentified by the ITS LT database except for Trichosporon mucoides whose ITS LT profile was identical to that of Trichosporon dermatis. In conclusion, yeast identification by the present SCGE ITS LT assay is a fast, reproducible and accurate alternative for the identification of clinically important yeasts with the exception of Trichosporon species. PMID:27105313

  11. Surveying expression level polymorphism and single-feature polymorphism in near-isogenic wheat lines differing for the Yr5 stripe rust resistance locus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA polymorphisms are valuable for several applications including genotyping, molecular mapping and marker-assisted selection. The Affymetrix Wheat GeneChip was used to survey expression level polymorphisms (ELPs) and single-feature polymorphisms (SFPs) between two near-isogenic wheat genotypes (BC7...

  12. Surveying expression level polymorphism and single-feature polymorphism in near-isogenic wheat lines differing for the Yr5 stripe rust resistance locus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA polymorphisms are valuable for several applications including genotyping, molecular mapping and marker-assisted selection. The Affymetrix Wheat GeneChip was used to survey expression level polymorphisms (ELPs) and single-feature polymorphisms (SFPs) between two near-isogenic wheat genotypes (BC...

  13. Microarray-Based Genetic Mapping Using Soybean Near-Isogenic Lines and Generation of SNP Markers in the Rag1 Aphid-Resistance Interval

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A strategy using near-isogenic lines (NILs) and Affymetrix Soybean GeneChip microarrays was employed to identify genetic markers closely linked to the soybean aphid [Aphis glycines Matsumura (Hemiptera: Aphididae)] resistance gene Rag1 in soybean [Glycine max (L.) Merr.]. Genomic DNA from the aphid ...

  14. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes*

    PubMed Central

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-01-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China. PMID:26642182

  15. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes.

    PubMed

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-12-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China. PMID:26642182

  16. DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and {gamma}-rays

    SciTech Connect

    Kimura, Shinzo; Ishidou, Emi; Kurita, Sakiko; Suzuki, Yoshiteru; Shibato, Junko; Rakwal, Randeep . E-mail: rakwal-68@aist.go.jp; Iwahashi, Hitoshi

    2006-07-21

    Ionizing radiation (IR) is the most enigmatic of genotoxic stress inducers in our environment that has been around from the eons of time. IR is generally considered harmful, and has been the subject of numerous studies, mostly looking at the DNA damaging effects in cells and the repair mechanisms therein. Moreover, few studies have focused on large-scale identification of cellular responses to IR, and to this end, we describe here an initial study on the transcriptional responses of the unicellular genome model, yeast (Saccharomyces cerevisiae strain S288C), by cDNA microarray. The effect of two different IR, X-rays, and gamma ({gamma})-rays, was investigated by irradiating the yeast cells cultured in YPD medium with 50 Gy doses of X- and {gamma}-rays, followed by resuspension of the cells in YPD for time-course experiments. The samples were collected for microarray analysis at 20, 40, and 80 min after irradiation. Microarray analysis revealed a time-course transcriptional profile of changed gene expressions. Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and {gamma}-rays). Similarly, for X- and {gamma}-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. This study provides for the first time a snapshot of the genome-wide mRNA expression profiles in X- and {gamma}-ray post-irradiated yeast cells and comparatively interprets/discusses the changed gene functional categories as effects of these two radiations vis-a-vis their energy levels.

  17. Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha.

    PubMed

    Klabunde, J; Diesel, A; Waschk, D; Gellissen, G; Hollenberg, C P; Suckow, M

    2002-05-01

    We have investigated the methylotrophic yeast Hansenula polymorpha as a host for the co-integration and expression of multiple heterologous genes using an rDNA integration approach. The ribosomal DNA (rDNA) of H. polymorpha was found to consist of a single rDNA cluster of about 50-60 repeats of an 8-kb unit located on chromosome II. A 2.4-kb segment of H. polymorpha rDNA encompassing parts of the 25S, the complete 5S and the non-transcribed spacer region between 25S and 18S rDNA was isolated and inserted into conventional integrative H. polymorpha plasmids harboring the Saccharomyces- cerevisiae-derived URA3 gene for selection. These rDNA plasmids integrated homologously into the rDNA repeats of a H. polymorpha (odc1) host as several independent clusters. Anticipating that this mode of multiple-cluster integration could be used for the simultaneous integration of several distinct rDNA plasmids, the host strain was co-transformed with a mixture of up to three different plasmids, all bearing the same URA3 selection marker. Transformations indeed resulted in mitotically stable strains harboring one, two, or all three plasmids integrated into the rDNA. The overall copy number of the plasmids integrated did not exceed the number of rDNA repeats present in the untransformed host strain, irrespective of the number of different plasmids involved. Strains harboring different plasmids co-expressed the introduced genes, resulting in functional proteins. Thus, this approach provides a new and attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios. PMID:12021801

  18. Paramagnetic cobalt as a probe of the orientation of an accessory DNA-binding region of the yeast ADR1 zinc-finger protein.

    PubMed

    Schmiedeskamp, M; Klevit, R E

    1997-11-18

    The minimal DNA-binding domain of the yeast ADR1 transcription factor consists of two Cys2-His2 zinc fingers and an additional 20 residues N-terminal and proximal to the fingers. The accessory sequence likely plays a role in contacting DNA. Paramagnetic cobalt was incorporated into the fingers of an ADR1 DNA-binding construct (ADR1z) to serve as a probe of the proximity of the accessory sequence to the zinc fingers. NMR signals from the accessory region are not perturbed by cobalt incorporation. Previous studies showed that this region is random coil in the ADR1z construct in the absence of DNA; it does not adopt a fixed orientation with respect to the cobalt sites. In contrast, many residues of the accessory region are perturbed by cobalt in the DNA-bound form of the protein, suggesting this region becomes constrained. This observation agrees with previous results showing a disorder-to-order transition for the accessory region upon DNA binding. Furthermore, these results indicate that the accessory region lies close to the fingers in the protein-DNA complex. This region thus does not extend along the DNA away from the zinc fingers; it more likely binds the same stretch of DNA contacted by the zinc fingers. Comparison to the behavior of other zinc-finger proteins that utilize an accessory DNA-binding sequence suggested that the region of ADR1 proximal to the zinc fingers might form an alpha-helix. Analysis of sequential NOEs in the accessory region of DNA-bound ADR1z reveals no helical structure. PMID:9369471

  19. A Functional Yeast Survival Screen of Tumor-Derived cDNA Libraries Designed to Identify Anti-Apoptotic Mammalian Oncogenes

    PubMed Central

    Melzer, Inga Maria; Moser, Julia; Siele, Dagmar; Köhl, Ulrike; Rieker, Ralf Joachim; Wachter, David Lukas; Agaimy, Abbas; Herpel, Esther; Baumgarten, Peter; Mittelbronn, Michel; Rakel, Stefanie; Kögel, Donat; Böhm, Stefanie; Gutschner, Tony; Diederichs, Sven; Zörnig, Martin

    2013-01-01

    Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems. PMID:23717670

  20. Apn1 AP-endonuclease is essential for the repair of oxidatively damaged DNA bases in yeast frataxin-deficient cells

    PubMed Central

    Lefevre, Sophie; Brossas, Caroline; Auchère, Françoise; Boggetto, Nicole; Camadro, Jean-Michel; Santos, Renata

    2012-01-01

    Frataxin deficiency results in mitochondrial dysfunction and oxidative stress and it is the cause of the hereditary neurodegenerative disease Friedreich ataxia (FA). Here, we present evidence that one of the pleiotropic effects of oxidative stress in frataxin-deficient yeast cells (Δyfh1 mutant) is damage to nuclear DNA and that repair requires the Apn1 AP-endonuclease of the base excision repair pathway. Major phenotypes of Δyfh1 cells are respiratory deficit, disturbed iron homeostasis and sensitivity to oxidants. These phenotypes are weak or absent under anaerobiosis. We show here that exposure of anaerobically grown Δyfh1 cells to oxygen leads to down-regulation of antioxidant defenses, increase in reactive oxygen species, delay in G1- and S-phases of the cell cycle and damage to mitochondrial and nuclear DNA. Nuclear DNA lesions in Δyfh1 cells are primarily caused by oxidized bases and single-strand breaks that can be detected 15–30 min after oxygen exposition. The Apn1 enzyme is essential for the repair of the DNA lesions in Δyfh1 cells. Compared with Δyfh1, the double Δyfh1Δapn1 mutant shows growth impairment, increased mutagenesis and extreme sensitivity to H2O2. On the contrary, overexpression of the APN1 gene in Δyfh1 cells decreases spontaneous and induced mutagenesis. Our results show that frataxin deficiency in yeast cells leads to increased DNA base oxidation and requirement of Apn1 for repair, suggesting that DNA damage and repair could be important features in FA disease progression. PMID:22706278

  1. The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants.

    PubMed

    Hipp, Katharina; Rau, Peter; Schäfer, Benjamin; Gronenborn, Bruno; Jeske, Holger

    2014-08-01

    Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clones prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast. PMID:24999043

  2. The Protein Level of Rev1, a TLS Polymerase in Fission Yeast, Is Strictly Regulated during the Cell Cycle and after DNA Damage

    PubMed Central

    Uchiyama, Masashi; Terunuma, Junko; Hanaoka, Fumio

    2015-01-01

    Translesion DNA synthesis provides an alternative DNA replication mechanism when template DNA is damaged. In fission yeast, Eso1 (polη), Kpa1/DinB (polκ), Rev1, and Polζ (a complex of Rev3 and Rev7) have been identified as translesion synthesis polymerases. The enzymatic characteristics and protein-protein interactions of these polymerases have been intensively characterized; however, how these proteins are regulated during the cell cycle remains unclear. Therefore, we examined the cell cycle oscillation of translesion polymerases. Interestingly, the protein levels of Rev1 peaked during G1 phase and then decreased dramatically at the entry of S phase; this regulation was dependent on the proteasome. Temperature-sensitive proteasome mutants, such as mts2-U31 and mts3-U32, stabilized Rev1 protein when the temperature was shifted to the restrictive condition. In addition, deletion of pop1 or pop2, subunits of SCF ubiquitin ligase complexes, upregulated Rev1 protein levels. Besides these effects during the cell cycle, we also observed upregulation of Rev1 protein upon DNA damage. This upregulation was abolished when rad3, a checkpoint protein, was deleted or when the Rev1 promoter was replaced with a constitutive promoter. From these results, we hypothesize that translesion DNA synthesis is strictly controlled through Rev1 protein levels in order to avoid unwanted mutagenesis. PMID:26147350

  3. Determination of fruit origin by using 26S rDNA fingerprinting of yeast communities by PCR-DGGE: preliminary application to Physalis fruits from Egypt.

    PubMed

    El Sheikha, Aly Farag; Condur, Ana; Métayer, Isabelle; Nguyen, Doan Duy Le; Loiseau, Gérard; Montet, Didier

    2009-10-01

    The determination of geographical origin is a demand of the traceability system of import-export food products. One hypothesis for tracing the source of a product is by global analysis of the microbial communities of the food and statistical linkage of this analysis to the geographical origin of the food. For this purpose, a molecular technique employing 26S rDNA profiles generated by PCR-DGGE was used to detect the variation in yeast community structures of three species of Physalis fruit (Physalis ixocarpa Brat, Physalis pubescens L, Physalis pruinosa L) from four Egyptian regions (Qalyoubia, Minufiya, Beheira and Alexandria Governments). When the 26S rDNA profiles were analysed by multivariate analysis, distinct microbial communities were detected. The band profiles of Physalis yeasts from different Governments were specific for each location and could be used as a bar code to discriminate the origin of the fruits. This method is a new traceability tool which provides fruit products with a unique biological bar code and makes it possible to trace back the fruits to their original location. PMID:19784935

  4. Analysis of the DNA-Binding Activities of the Arabidopsis R2R3-MYB Transcription Factor Family by One-Hybrid Experiments in Yeast

    PubMed Central

    Kelemen, Zsolt; Sebastian, Alvaro; Xu, Wenjia; Grain, Damaris; Salsac, Fabien; Avon, Alexandra; Berger, Nathalie; Tran, Joseph; Dubreucq, Bertrand; Lurin, Claire; Lepiniec, Loïc; Contreras-Moreira, Bruno; Dubos, Christian

    2015-01-01

    The control of growth and development of all living organisms is a complex and dynamic process that requires the harmonious expression of numerous genes. Gene expression is mainly controlled by the activity of sequence-specific DNA binding proteins called transcription factors (TFs). Amongst the various classes of eukaryotic TFs, the MYB superfamily is one of the largest and most diverse, and it has considerably expanded in the plant kingdom. R2R3-MYBs have been extensively studied over the last 15 years. However, DNA-binding specificity has been characterized for only a small subset of these proteins. Therefore, one of the remaining challenges is the exhaustive characterization of the DNA-binding specificity of all R2R3-MYB proteins. In this study, we have developed a library of Arabidopsis thaliana R2R3-MYB open reading frames, whose DNA-binding activities were assayed in vivo (yeast one-hybrid experiments) with a pool of selected cis-regulatory elements. Altogether 1904 interactions were assayed leading to the discovery of specific patterns of interactions between the various R2R3-MYB subgroups and their DNA target sequences and to the identification of key features that govern these interactions. The present work provides a comprehensive in vivo analysis of R2R3-MYB binding activities that should help in predicting new DNA motifs and identifying new putative target genes for each member of this very large family of TFs. In a broader perspective, the generated data will help to better understand how TF interact with their target DNA sequences. PMID:26484765

  5. cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases.

    PubMed Central

    Seidah, N G; Hamelin, J; Mamarbachi, M; Dong, W; Tardos, H; Mbikay, M; Chretien, M; Day, R

    1996-01-01

    By using reverse transcription-coupled PCR on rat anterior pituitary RNA, we isolated a 285-bp cDNA coding for a novel subtilisin/kexin-like protein convertase (PC), called rat (r) PC7. By screening rat spleen and PC12 cell lambda gt11 cDNA libraries, we obtained a composite 3.5-kb full-length cDNA sequence of rPC7. The open reading frame codes for a prepro-PC with a 36-amino acid signal peptide, a 104-amino acid prosegment ending with a cleavable RAKR sequence, and a 747-amino acid type I membrane-bound glycoprotein, representing the mature form of this serine proteinase. Phylogenetic analysis suggests that PC7 represents the most divergent enzyme of the mammalian convertase family and that it is the closest member to the yeast convertases krp and kexin. Northern blot analyses demonstrated a widespread expression with the richest source of rPC7 mRNA being the colon and lymphoid-associated tissues. In situ hybridization revealed a distinctive tissue distribution that sometimes overlaps with that of furin, suggesting that PC7 has widespread proteolytic functions. The gene for PC7 (Pcsk7) was mapped to mouse chromosome 9 by linkage analysis of an interspecific backcross DNA panel. Images Fig. 3 Fig. 4 Fig. 5 PMID:8622945

  6. The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo

    PubMed Central

    Henikoff, Steven; Ramachandran, Srinivas; Krassovsky, Kristina; Bryson, Terri D; Codomo, Christine A; Brogaard, Kristin; Wang, Ji-Ping; Henikoff, Jorja G

    2014-01-01

    In budding yeast, a single cenH3 (Cse4) nucleosome occupies the ∼120-bp functional centromere, however conflicting structural models for the particle have been proposed. To resolve this controversy, we have applied H4S47C-anchored cleavage mapping, which reveals the precise position of histone H4 in every nucleosome in the genome. We find that cleavage patterns at centromeres are unique within the genome and are incompatible with symmetrical structures, including octameric nucleosomes and (Cse4/H4)2 tetrasomes. Centromere cleavage patterns are compatible with a precisely positioned core structure, one in which each of the 16 yeast centromeres is occupied by oppositely oriented Cse4/H4/H2A/H2B hemisomes in two rotational phases within the population. Centromere-specific hemisomes are also inferred from distances observed between closely-spaced H4 cleavages, as predicted from structural modeling. Our results indicate that the orientation and rotational position of the stable hemisome at each yeast centromere is not specified by the functional centromere sequence. DOI: http://dx.doi.org/10.7554/eLife.01861.001 PMID:24737863

  7. Negative superhelicity promotes ATP-dependent binding of yeast RAD3 protein to ultraviolet-damaged DNA.

    PubMed

    Sung, P; Watkins, J F; Prakash, L; Prakash, S

    1994-03-18

    The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of UV-damaged DNA and is essential for cell viability. Remarkable homology exists between RAD3 and the human excision repair gene XPD, whose mutational inactivation underlies the cancer-prone disorder in xeroderma pigmentosum group D patients. Our previous work demonstrated that RAD3-encoded protein contains a DNA helicase activity. Here, we show that RAD3 binds preferentially to UV-damaged DNA over nondamaged DNA. Removal of pyrimidine dimers from damaged DNA by enzymatic photoreactivation does not affect binding, suggesting an affinity of RAD3 for pyrimidine (6-4) pyrimidone photoproducts. Damage-specific binding by RAD3 is strongly dependent on ATP and on the degree of negative superhelicity in DNA. The requirement of superhelicity in damage binding may target RAD3 to regions of DNA undergoing transcription, resulting in the preferential repair of these regions. The rad3 Arg-48 mutant protein, which lacks the DNA helicase activity, also binds UV-damaged DNA preferentially, indicating that DNA helicase and damage binding are two distinct and separable functional entities in RAD3. PMID:8132553

  8. Epidemiologic Study of Malassezia Yeasts in Seborrheic Dermatitis Patients by the Analysis of 26S rDNA PCR-RFLP

    PubMed Central

    Oh, Byung Ho; Choe, Yong Beom; Ahn, Kyu Joong

    2010-01-01

    Background This case-control study concerns a molecular biological method based on the data gathered from a group of Korean subjects to examine the distribution of Malassezia yeasts in seborrheic dermatitis (SD) patients. Cultures for Malassezia yeasts were taken from the foreheads, cheeks and chests of 60 patients with SD and in 60 healthy controls of equivalent age. Objective The purpose of this study is to identify the relationship between certain species of Malassezia and SD. This was done by analyzing the differences in the distribution of Malassezia species in terms of age and body parts of the host with healthy controls. Methods 26S rDNA PCR-RFLP, a fast and accurate molecular biological method, was used to overcome the limits of morphological and biochemical methods. Results The positive Malassezia culture rate was 51.7% in patients with SD, which was lower than that of healthy adults (63.9%). M. restricta was dominant in patients with SD (19.5%). Likewise, M. restricta was identified as a common species (20.5%) in healthy controls. In the ages 31~40, M. restricta was found to be the most common species (31.6%) among SD patients. Conclusion According to the results of the study, the most frequently isolated species was M. restricta (19.5%) in patients with SD. There was no statistically significant difference in the distribution of Malassezia species between the SD patients and healthy control groups. PMID:20548904

  9. Yeast Infections

    MedlinePlus

    ... antibiotics, it can multiply and cause an infection. Yeast infections affect different parts of the body in different ways: Thrush is a yeast infection that causes white patches in your mouth Candida ...

  10. Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast.

    PubMed

    Shiosaki, K; Takata, K; Omichi, K; Tomita, N; Horii, A; Ogawa, M; Matsubara, K

    1990-05-14

    A novel amylase gene (amy3) that differs in nucleotide sequence from salivary amylase gene (amy1) and pancreatic amylase gene (amy2) has been described [Tomita et al., Gene 76 (1989) 11-18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel alpha-amylase gene (amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes. PMID:2197187

  11. More than 10% of yeast genes are related to genome stability and influence cellular senescence via rDNA maintenance.

    PubMed

    Saka, Kimiko; Takahashi, Akihiro; Sasaki, Mariko; Kobayashi, Takehiko

    2016-05-19

    Genome instability triggers cellular senescence and is a common cause of cancer. The ribosomal RNA genes (rDNA), due to their repetitive structure, form a fragile site with frequent rearrangements. To identify eukaryotic factors that connect reduced genome stability to senescence we screened 4,876 strains of a Saccharomyces cerevisiae deletion library for aberrant rDNA and found 708 genes that contribute to its upkeep. 28 mutants caused abnormalities in non-rDNA chromosomes and among them 12 mutants have abnormalities both in rDNA and in non-rDNA chromosomes. Many mutated genes have not previously been implicated with genome maintenance nor their homologues with tumorigenesis in mammals. The link between rDNA state and senescence was broken after deletion of factors related with DNA polymerase ϵ. These mutations also suppressed the short lifespan phenotype of a sir2 mutant, suggesting a model in which molecular events at the heart of the replication fork induce abnormal rDNA recombination and are responsible for the emergence of an aging signal. PMID:26912831

  12. A yeast gene, MGS1, encoding a DNA-dependent AAA(+) ATPase is required to maintain genome stability.

    PubMed

    Hishida, T; Iwasaki, H; Ohno, T; Morishita, T; Shinagawa, H

    2001-07-17

    Changes in DNA superhelicity during DNA replication are mediated primarily by the activities of DNA helicases and topoisomerases. If these activities are defective, the progression of the replication fork can be hindered or blocked, which can lead to double-strand breaks, elevated recombination in regions of repeated DNA, and genome instability. Hereditary diseases like Werner's and Bloom's Syndromes are caused by defects in DNA helicases, and these diseases are associated with genome instability and carcinogenesis in humans. Here we report a Saccharomyces cerevisiae gene, MGS1 (Maintenance of Genome Stability 1), which encodes a protein belonging to the AAA(+) class of ATPases, and whose central region is similar to Escherichia coli RuvB, a Holliday junction branch migration motor protein. The Mgs1 orthologues are highly conserved in prokaryotes and eukaryotes. The Mgs1 protein possesses DNA-dependent ATPase and single-strand DNA annealing activities. An mgs1 deletion mutant has an elevated rate of mitotic recombination, which causes genome instability. The mgs1 mutation is synergistic with a mutation in top3 (encoding topoisomerase III), and the double mutant exhibits severe growth defects and markedly increased genome instability. In contrast to the mgs1 mutation, a mutation in the sgs1 gene encoding a DNA helicase homologous to the Werner and Bloom helicases suppresses both the growth defect and the increased genome instability of the top3 mutant. Therefore, evolutionarily conserved Mgs1 may play a role together with RecQ family helicases and DNA topoisomerases in maintaining proper DNA topology, which is essential for genome stability. PMID:11459965

  13. Yeast high mobility group protein HMO1 stabilizes chromatin and is evicted during repair of DNA double strand breaks

    PubMed Central

    Panday, Arvind; Xiao, LiJuan; Grove, Anne

    2015-01-01

    DNA is packaged into condensed chromatin fibers by association with histones and architectural proteins such as high mobility group (HMGB) proteins. However, this DNA packaging reduces accessibility of enzymes that act on DNA, such as proteins that process DNA after double strand breaks (DSBs). Chromatin remodeling overcomes this barrier. We show here that the Saccharomyces cerevisiae HMGB protein HMO1 stabilizes chromatin as evidenced by faster chromatin remodeling in its absence. HMO1 was evicted along with core histones during repair of DSBs, and chromatin remodeling events such as histone H2A phosphorylation and H3 eviction were faster in absence of HMO1. The facilitated chromatin remodeling in turn correlated with more efficient DNA resection and recruitment of repair proteins; for example, inward translocation of the DNA-end-binding protein Ku was faster in absence of HMO1. This chromatin stabilization requires the lysine-rich C-terminal extension of HMO1 as truncation of the HMO1 C-terminal tail phenocopies hmo1 deletion. Since this is reminiscent of the need for the basic C-terminal domain of mammalian histone H1 in chromatin compaction, we speculate that HMO1 promotes chromatin stability by DNA bending and compaction imposed by its lysine-rich domain and that it must be evicted along with core histones for efficient DSB repair. PMID:25979266

  14. A novel cis-acting element required for DNA damage-inducible expression of yeast DIN7

    SciTech Connect

    Yoshitani, Ayako; Yoshida, Minoru; Ling Feng

    2008-01-04

    Din7 is a DNA damage-inducible mitochondrial nuclease that modulates the stability of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. How DIN7 gene expression is regulated, however, has remained largely unclear. Using promoter sequence alignment, we found a highly conserved 19-bp sequence in the promoter regions of DIN7 and NTG1, which encodes an oxidative stress-inducible base-excision-repair enzyme. Deletion of the 19-bp sequence markedly reduced the hydroxyurea (HU)-enhanced DIN7 promoter activity. In addition, nuclear fractions prepared from HU-treated cells were used in in vitro band shift assays to reveal the presence of currently unidentified trans-acting factor(s) that preferentially bound to the 19-bp region. These results suggest that the 19-bp sequence is a novel cis-acting element that is required for the regulation of DIN7 expression in response to HU-induced DNA damage.

  15. C(m)CGG methylation-independent parent-of-origin effects on genome-wide transcript levels in isogenic reciprocal F1 triploid plants.

    PubMed

    Donoghue, Mark T A; Fort, Antoine; Clifton, Rachel; Zhang, Xu; McKeown, Peter C; Voigt-Zielinksi, M L; Borevitz, Justin O; Spillane, Charles

    2014-01-01

    Triploid F1 hybrids generated via reciprocal interploidy crosses between genetically distinct parental plants can display parent-of-origin effects on gene expression or phenotypes. Reciprocal triploid F1 isogenic plants generated from interploidy crosses in the same genetic background allow investigation on parent-of-origin-specific (parental) genome-dosage effects without confounding effects of hybridity involving heterozygous mutations. Whole-genome transcriptome profiling was conducted on reciprocal F1 isogenic triploid (3x) seedlings of A. thaliana. The genetically identical reciprocal 3x genotypes had either an excess of maternally inherited 3x(m) or paternally inherited 3x(p) genomes. We identify a major parent-of-origin-dependent genome-dosage effect on transcript levels, whereby 602 genes exhibit differential expression between the reciprocal F1 triploids. In addition, using methylation-sensitive DNA tiling arrays, constitutive and polymorphic CG DNA methylation patterns at CCGG sites were analysed, which revealed that paternal-excess F1 triploid seedling C(m)CGG sites are overall hypermethylated. However, no correlation exists between C(m)CGG methylation polymorphisms and transcriptome dysregulation between the isogenic reciprocal F1 triploids. Overall, our study indicates that parental genome-dosage effects on the transcriptome levels occur in paternal-excess triploids, which are independent of C(m)CGG methylation polymorphisms. Such findings have implications for understanding parental effects and genome-dosage effects on gene expression and phenotypes in polyploid plants. PMID:24212467

  16. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    SciTech Connect

    Pang, Jinsong; Dong, Mingyue; Li, Ning; Zhao, Yanli; Liu, Bao

    2013-03-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  17. Use of a Ring Chromosome and Pulsed-Field Gels to Study Interhomolog Recombination, Double-Strand DNA Breaks and Sister-Chromatid Exchange in Yeast

    PubMed Central

    Game, J. C.; Sitney, K. C.; Cook, V. E.; Mortimer, R. K.

    1989-01-01

    We describe a system that uses pulsed-field gels for the physical detection of recombinant DNA molecules, double-strand DNA breaks (DSB) and sister-chromatid exchange in the yeast Saccharomyces cerevisiae. The system makes use of a circular variant of chromosome III (Chr. III). Meiotic recombination between this ring chromosome and a linear homolog produces new molecules of sizes distinguishable on gels from either parental molecule. We demonstrate that these recombinant molecules are not present either in strains with two linear Chr. III molecules or in rad50 mutants, which are defective in meiotic recombination. In conjunction with the molecular endpoints, we present data on the timing of commitment to meiotic recombination scored genetically. We have used x-rays to linearize circular Chr. III, both to develop a sensitive method for measuring frequency of DSB and as a means of detecting double-sized circles originating in part from sister-chromatid exchange, which we find to be frequent during meiosis. PMID:2693206

  18. Yeast DNA ligase IV mutations reveal a nonhomologous end joining function of BRCT1 distinct from XRCC4/Lif1 binding

    PubMed Central

    Chiruvella, Kishore K.; Renard, Brian M.; Birkeland, Shanda R.; Sunder, Sham; Liang, Zhuobin; Wilson, Thomas E.

    2014-01-01

    LIG4/Dnl4 is the DNA ligase that (re)joins DNA double-strand breaks (DSBs) via nonhomologous end joining (NHEJ), an activity supported by binding of its tandem BRCT domains to the ligase accessory protein XRCC4/Lif1. We screened a panel of 88 distinct ligase mutants to explore the structure-function relationships of the yeast Dnl4 BRCT domains and inter-BRCT linker in NHEJ. Screen results suggested two distinct classes of BRCT mutations with differential effects on Lif1 interaction as compared to NHEJ completion. Validated constructs confirmed that D800K and GG(868:869)AA mutations, which target the Lif1 binding interface, showed a severely defective Dnl4-Lif1 interaction but a less consistent and often small decrease in NHEJ activity in some assays, as well as nearly normal levels of Dnl4 accumulation at DSBs. In contrast, mutants K742A and KTT(742:744)ATA, which target the β3-α2 region of the first BRCT domain, substantially decreased NHEJ function commensurate with a large defect in Dnl4 recruitment to DSBs, despite a comparatively greater preservation of the Lif1 interaction. Together, these separation-of-function mutants indicate that Dnl4 BRCT1 supports DSB recruitment and NHEJ in a manner distinct from Lif1 binding and reveal a complexity of Dnl4 BRCT domain functions in support of stable DSB association. PMID:25457772

  19. A novel DNA binding motif for yeast zinc cluster proteins: the Leu3p and Pdr3p transcriptional activators recognize everted repeats.

    PubMed Central

    Hellauer, K; Rochon, M H; Turcotte, B

    1996-01-01

    The Gal4, Put3, and Ppr1 yeast zinc cluster proteins bind as homodimers to DNA sequences composed of palindromic CGG triplets. Spacing between the triplets specifies the target site for a given zinc cluster protein. In addition, Hap1p, another zinc cluster protein, also recognizes CGG triplets but only when oriented as a direct repeat. Unexpectedly, our results show that Leu3p, another member of this family, also recognizes CGG triplets but oriented in opposite directions and spaced by 4 nucleotides (an everted repeat or inverted palindrome: CCG-N4-CGG). This constitutes a novel DNA motif for zinc cluster proteins. Moreover, the presence of this motif was shown to be essential for in vivo activation by Leu3p of a minimal reporter containing one copy of a target site for this activator. We also provide evidence that another member of this family, Pdr3p, binds to an everted repeat spaced by 0 nucleotides (CCGCGG). Thus, our results show that three CGG motifs are used by members of the zinc cluster family: palindromes, direct repeats, and everted repeats. PMID:8887639

  20. Use of a ring chromosome and pulsed-field gels to study interhomolog recombination, double-strand DNA breaks and sister-chromatid exchange in yeast

    SciTech Connect

    Game, J.C. ); Sitney, K.C.; Cook, V.E.; Mortimer, R.K. )

    1989-12-01

    The authors describe a system that uses pulsed-field gels for the physical detection of recombinant DNA molecules, double-strand DNA breaks (DSB) and sister-chromatid exchange in the yeast Saccharomyces cerevisiae. The system makes use of a circular variant of chromosome II (Chr. III). Meiotic recombination between this ring chromosome and a linear homolog produces new molecules of sizes distinguishable on gels from either parental molecule. They demonstrate that these recombinant molecules are not present either in strains with two linear Chr. III molecules or in rad50 mutants, which are defective in meiotic recombination. In conjunction with the molecular endpoints. They present data on the timing of commitment to meiotic recombination scored genetically. They have used x-rays to linearize circular Chr. III, both to develop a sensitive method for measuring frequency of DSB and as a means of detecting double-size circles originating in part from sister-chromatid exchange, which they find to be frequent during meiosis.

  1. A unique nucleosome arrangement, maintained actively by chromatin remodelers facilitates transcription of yeast tRNA genes

    PubMed Central

    2013-01-01

    Background RNA polymerase (pol) III transcribes a unique class of genes with intra-genic promoters and high transcriptional activity. The major contributors to the pol III transcriptome, tRNAs genes are found scattered on all chromosomes of yeast. A prototype tDNA of <150 bp length, is generally considered nucleosome-free while some pol III-transcribed genes have been shown to have nucleosome-positioning properties. Results Using high resolution ChIP-chip and ChIP-seq methods, we found several unique features associated with nucleosome profiles on all tRNA genes of budding yeast, not seen on nucleosome-dense counterparts in fission yeast and resting human CD4+ T cells. The nucleosome-free region (NFR) on all but three yeast tDNAs is found bordered by an upstream (US) nucleosome strongly positioned at −140 bp position and a downstream (DS) nucleosome at variable positions with respect to the gene terminator. Perturbation in this nucleosomal arrangement interferes with the tRNA production. Three different chromatin remodelers generate and maintain the NFR by targeting different gene regions. Isw1 localizes to the gene body and makes it nucleosome-depleted, Isw2 maintains periodicity in the upstream nucleosomal array, while RSC targets the downstream nucleosome. Direct communication of pol III with RSC serves as a stress-sensory mechanism for these genes. In its absence, the downstream nucleosome moves towards the gene terminator. Levels of tRNAs from different families are found to vary considerably as different pol III levels are seen even on isogenes within a family. Pol III levels show negative correlation with the nucleosome occupancies on different genes. Conclusions Budding yeast tRNA genes maintain an open chromatin structure, which is not due to sequence-directed nucleosome positioning or high transcription activity of genes. Unlike 5′ NFR on pol II-transcribed genes, the tDNA NFR, which facilitates tDNA transcription, results from action of chromatin

  2. A single zinc finger optimizes the DNA interactions of the nucleocapsid protein of the yeast retrotransposon Ty3

    PubMed Central

    Chaurasiya, Kathy R.; Geertsema, Hylkje; Cristofari, Gaël; Darlix, Jean-Luc; Williams, Mark C.

    2012-01-01

    Reverse transcription in retroviruses and retrotransposons requires nucleic acid chaperones, which drive the rearrangement of nucleic acid conformation. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein have been extensively studied, and nucleic acid aggregation, duplex destabilization and rapid binding kinetics have been identified as major components of its activity. However, the properties of other nucleic acid chaperone proteins, such as retrotransposon Ty3 NC, a likely ancestor of HIV-1 NC, are not well understood. In addition, it is unclear whether a single zinc finger is sufficient to optimize the properties characteristic of HIV-1 NC. We used single-molecule DNA stretching as a method for detailed characterization of Ty3 NC chaperone activity. We found that wild type Ty3 NC aggregates single- and double-stranded DNA, weakly stabilizes dsDNA, and exhibits rapid binding kinetics. Single-molecule studies in the presence of Ty3 NC mutants show that the N-terminal basic residues and the unique zinc finger at the C-terminus are required for optimum chaperone activity in this system. While the single zinc finger is capable of optimizing Ty3 NC's DNA interaction kinetics, two zinc fingers may be necessary in order to facilitate the DNA destabilization exhibited by HIV-1 NC. PMID:21917850

  3. A single zinc finger optimizes the DNA interactions of the nucleocapsid protein of the yeast retrotransposon Ty3.

    PubMed

    Chaurasiya, Kathy R; Geertsema, Hylkje; Cristofari, Gaël; Darlix, Jean-Luc; Williams, Mark C

    2012-01-01

    Reverse transcription in retroviruses and retrotransposons requires nucleic acid chaperones, which drive the rearrangement of nucleic acid conformation. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein have been extensively studied, and nucleic acid aggregation, duplex destabilization and rapid binding kinetics have been identified as major components of its activity. However, the properties of other nucleic acid chaperone proteins, such as retrotransposon Ty3 NC, a likely ancestor of HIV-1 NC, are not well understood. In addition, it is unclear whether a single zinc finger is sufficient to optimize the properties characteristic of HIV-1 NC. We used single-molecule DNA stretching as a method for detailed characterization of Ty3 NC chaperone activity. We found that wild type Ty3 NC aggregates single- and double-stranded DNA, weakly stabilizes dsDNA, and exhibits rapid binding kinetics. Single-molecule studies in the presence of Ty3 NC mutants show that the N-terminal basic residues and the unique zinc finger at the C-terminus are required for optimum chaperone activity in this system. While the single zinc finger is capable of optimizing Ty3 NC's DNA interaction kinetics, two zinc fingers may be necessary in order to facilitate the DNA destabilization exhibited by HIV-1 NC. PMID:21917850

  4. Genomic Heterogeneity and Structural Variation in Soybean Near Isogenic Lines

    PubMed Central

    Stec, Adrian O.; Bhaskar, Pudota B.; Bolon, Yung-Tsi; Nolan, Rebecca; Shoemaker, Randy C.; Vance, Carroll P.; Stupar, Robert M.

    2013-01-01

    Near isogenic lines (NILs) are a critical genetic resource for the soybean research community. The ability to identify and characterize the genes driving the phenotypic differences between NILs is limited by the degree to which differential genetic introgressions can be resolved. Furthermore, the genetic heterogeneity extant among NIL sub-lines is an unaddressed research topic that might have implications for how genomic and phenotypic data from NILs are utilized. In this study, a recently developed high-resolution comparative genomic hybridization (CGH) platform was used to investigate the structure and diversity of genetic introgressions in two classical soybean NIL populations, respectively varying in protein content and iron deficiency chlorosis (IDC) susceptibility. There were three objectives: assess the capacity for CGH to resolve genomic introgressions, identify introgressions that are heterogeneous among NIL sub-lines, and associate heterogeneous introgressions with susceptibility to IDC. Using the CGH approach, introgression boundaries were refined and previously unknown introgressions were revealed. Furthermore, heterogeneous introgressions were identified within seven sub-lines of the IDC NIL “IsoClark.” This included three distinct introgression haplotypes linked to the major iron susceptible locus on chromosome 03. A phenotypic assessment of the seven sub-lines did not reveal any differences in IDC susceptibility, indicating that the genetic heterogeneity among the lines does not have a significant impact on the primary NIL phenotype. PMID:23630538

  5. Differential suppression of DNA repair deficiencies of Yeast rad50, mre11 and xrs2 mutants by EXO1 and TLC1 (the RNA component of telomerase).

    PubMed Central

    Lewis, L Kevin; Karthikeyan, G; Westmoreland, James W; Resnick, Michael A

    2002-01-01

    Rad50, Mre11, and Xrs2 form a nuclease complex that functions in both nonhomologous end-joining (NHEJ) and recombinational repair of DNA double-strand breaks (DSBs). A search for highly expressed cDNAs that suppress the DNA repair deficiency of rad50 mutants yielded multiple isolates of two genes: EXO1 and TLC1. Overexpression of EXO1 or TLC1 increased the resistance of rad50, mre11, and xrs2 mutants to ionizing radiation and MMS, but did not increase resistance in strains defective in recombination (rad51, rad52, rad54, rad59) or NHEJ only (yku70, sir4). Increased Exo1 or TLC1 RNA did not alter checkpoint responses or restore NHEJ proficiency, but DNA repair defects of yku70 and rad27 (fen) mutants were differentially suppressed by the two genes. Overexpression of Exo1, but not mutant proteins containing substitutions in the conserved nuclease domain, increased recombination and suppressed HO and EcoRI endonuclease-induced killing of rad50 strains. exo1 rad50 mutants lacking both nuclease activities exhibited a high proportion of enlarged, G2-arrested cells and displayed a synergistic decrease in DSB-induced plasmid:chromosome recombination. These results support a model in which the nuclease activity of the Rad50/Mre11/Xrs2 complex is required for recombinational repair, but not NHEJ. We suggest that the 5'-3' exo activity of Exo1 is able to substitute for Rad50/Mre11/Xrs2 in rescission of specific classes of DSB end structures. Gene-specific suppression by TLC1, which encodes the RNA subunit of the yeast telomerase complex, demonstrates that components of telomerase can also impact on DSB repair pathways. PMID:11805044

  6. Genomic Editing Tools to Model Human Diseases with Isogenic Pluripotent Stem Cells

    PubMed Central

    Kim, Huen Suk; Bernitz, Jeffrey M.; Lee, Dung-Fang

    2014-01-01

    Patient-specific induced pluripotent stem cells (iPSCs) are considered a versatile resource in the field of biomedicine. As iPSCs are generated on an individual basis, iPSCs may be the optimal cellular material to use for disease modeling, drug discovery, and the development of patient-specific cellular therapies. Recently, to gain an in-depth understanding of human pathologies, patient-specific iPSCs have been used to model human diseases with some iPSC-derived cells recapitulating pathological phenotypes in vitro. However, complex multigenic diseases generally have not resulted in concise conclusions regarding the underlying mechanisms of disease, in large part due to genetic variations between disease-state and control iPSCs. To circumvent this, the use of genomic editing tools to generate perfect isogenic controls is gaining momentum. To date, DNA binding domain-based zinc finger nucleases and transcription activator-like effector nucleases have been utilized to create genetically defined conditions in patient-specific iPSCs, with some examples leading to the successful identification of novel mechanisms of disease. As the feasibility and utility of genomic editing tools in iPSCs improve, along with the introduction of the clustered regularly interspaced short palindromic repeat system, understanding the features and limitations of genomic editing tools and their applications to iPSC technology is critical to expending the field of human disease modeling. PMID:25075441

  7. Dpb11 Controls the Association between DNA Polymerases α and ɛ and the Autonomously Replicating Sequence Region of Budding Yeast

    PubMed Central

    Masumoto, Hiroshi; Sugino, Akio; Araki, Hiroyuki

    2000-01-01

    Dpb11 is required for chromosomal DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. Here, we report detection of a physical complex containing Dpb11 and DNA polymerase ɛ (Dpb11-Polɛ complex). During the S phase of the cell cycle, Dpb11 associated preferentially with DNA fragments containing autonomously replicating sequences (ARSs), at the same time as Polɛ associated with these fragments. Association of Dpb11 and Polɛ with these fragments was mutually dependent, suggesting that the Dpb11-Polɛ complex associates with the ARS. Moreover, Dpb11 was required for the association of Polα-primase with the fragments. Thus, it seems likely that association of the Dpb11-Polɛ complex with the ARS fragments is required for the association of the Polα-primase complex. Hydroxyurea inhibits late-origin firing in S. cerevisiae, and the checkpoint genes, RAD53 and MEC1, are involved in this inhibition. In the presence of hydroxyurea at temperatures permissive for cell growth, Polɛ in dpb11-1 cells associated with early- and late-origin fragments. In wild-type cells, however, it associated only with early-origin fragments. This indicates that Dpb11 may also be involved in the regulation of late-origin firing. Overall, these results suggest that Dpb11 controls the association between DNA polymerases α and ɛ and the ARS. PMID:10733584

  8. The versatile thymine DNA-glycosylase: a comparative characterization of the human, Drosophila and fission yeast orthologs.

    PubMed

    Hardeland, Ulrike; Bentele, Marc; Jiricny, Josef; Schär, Primo

    2003-05-01

    Human thymine-DNA glycosylase (TDG) is well known to excise thymine and uracil from G.T and G.U mismatches, respectively, and was therefore proposed to play a central role in the cellular defense against genetic mutation through spontaneous deamination of 5-methylcytosine and cytosine. In this study, we characterized two newly discovered orthologs of TDG, the Drosophila melanogaster Thd1p and the Schizosaccharomyces pombe Thp1p proteins, with an objective to address the function of this subfamily of uracil-DNA glycosylases from an evolutionary perspective. A systematic biochemical comparison of both enzymes with human TDG revealed a number of biologically significant facts. (i) All eukaryotic TDG orthologs have broad and species-specific substrate spectra that include a variety of damaged pyrimidine and purine bases; (ii) the common most efficiently processed substrates of all are uracil and 3,N4- ethenocytosine opposite guanine and 5-fluorouracil in any double-stranded DNA context; (iii) 5-methylcytosine and thymine derivatives are processed with an appreciable efficiency only by the human and the Drosophila enzymes; (iv) none of the proteins is able to hydrolyze a non-damaged 5'-methylcytosine opposite G; and (v) the double strand and mismatch dependency of the enzymes varies with the substrate and is not a stringent feature of this subfamily of DNA glycosylases. These findings advance our current view on the role of TDG proteins and document that they have evolved with high structural flexibility to counter a broad range of DNA base damage in accordance with the specific needs of individual species. PMID:12711670

  9. Uniparental Inheritance of Mitochondrial Genes in Yeast: Dependence on Input Bias of Mitochondrial DNA and Preliminary Investigations of the Mechanism

    PubMed Central

    Birky, C. William; Demko, Catherine A.; Perlman, Philip S.; Strausberg, Robert

    1978-01-01

    In Saccharomyces cerevisiae, previous studies on the inheritance of mitochondrial genes controlling antibiotic resistance have shown that some crosses produce a substantial number of uniparental zygotes , which transmit to their diploid progeny mitochondrial alleles from only one parent. In this paper, we show that uniparental zygotes are formed especially when one parent (majority parent) contributes substantially more mitochondrial DNA molecules to the zygote than does the other (minority) parent. Cellular contents of mitochondrial DNA (mtDNA) are increased in these experiments by treatment with cycloheximide, alpha-factor, or the uvsρ5 nuclear mutation. In such a biased cross, some zygotes are uniparental for mitochondrial alleles from the majority parent, and the frequency of such zygotes increases with increasing bias. In two- and three-factor crosses, the cap1, ery1, and oli1 loci behave coordinately, rather than independently; minority markers tend to be transmitted or lost as a unit, suggesting that the uniparental mechanism acts on entire mtDNA molecules rather than on individual loci. This rules out the possibility that uniparental inheritance can be explained by the conversion of minority markers to the majority alleles during recombination. Exceptions to the coordinate behavior of different loci can be explained by marker rescue via recombination. Uniparental inheritance is largely independent of the position of buds on the zygote. We conclude that it is due to the failure of minority markers to replicate in some zygotes, possibly involving the rapid enzymatic destruction of such markers. We have considered two general classes of mechanisms: (1) random selection of molecules for replication, as for example by competition for replicating sites on a membrane; and (2) differential marking of mtDNA molecules in the two parents, possibly by modification enzymes, followed by a mechanism that "counts" molecules and replicates only the majority type. These

  10. Detection of leucine-independent DNA site occupancy of the yeast Leu3p transcriptional activator in vivo.

    PubMed Central

    Kirkpatrick, C R; Schimmel, P

    1995-01-01

    The product of the Saccharomyces cerevisiae LEU3 gene, Leu3p, is a transcriptional activator which regulates leucine biosynthesis in response to intracellular levels of leucine through the biosynthetic intermediate alpha-isopropylmalate. We devised a novel assay to examine the DNA site occupancy of Leu3p under different growth conditions, using a reporter gene with internal Leu3p-binding sites. Expression of the reporter is inhibited by binding of nuclear Leu3p to these sites; inhibition is dependent on the presence of the sites in the reporter, on the integrity of the Leu3p DNA-binding domain, and, surprisingly, on the presence of a transcriptional activation domain in the inhibiting protein. By this assay, Leu3p was found to occupy its binding site under all conditions tested, including high and low levels of leucine and in the presence and absence of alpha-isopropylmalate. The localization of Leu3p to the nucleus was confirmed by immunofluorescence staining of cells expressing epitope-tagged Leu3p derivatives. We conclude that Leu3p regulates transcription in vivo without changing its intracellular localization and DNA site occupancy. PMID:7623798

  11. The structure and DNA-binding properties of Mgm101 from a yeast with a linear mitochondrial genome

    PubMed Central

    Pevala, Vladimír; Truban, Dominika; Bauer, Jacob A.; Košťan, Július; Kunová, Nina; Bellová, Jana; Brandstetter, Marlene; Marini, Victoria; Krejčí, Lumír; Tomáška, Ľubomír; Nosek, Jozef; Kutejová, Eva

    2016-01-01

    To study the mechanisms involved in the maintenance of a linear mitochondrial genome we investigated the biochemical properties of the recombination protein Mgm101 from Candida parapsilosis. We show that CpMgm101 complements defects associated with the Saccharomyces cerevisiae mgm101–1ts mutation and that it is present in both the nucleus and mitochondrial nucleoids of C. parapsilosis. Unlike its S. cerevisiae counterpart, CpMgm101 is associated with the entire nucleoid population and is able to bind to a broad range of DNA substrates in a non-sequence specific manner. CpMgm101 is also able to catalyze strand annealing and D-loop formation. CpMgm101 forms a roughly C-shaped trimer in solution according to SAXS. Electron microscopy of a complex of CpMgm101 with a model mitochondrial telomere revealed homogeneous, ring-shaped structures at the telomeric single-stranded overhangs. The DNA-binding properties of CpMgm101, together with its DNA recombination properties, suggest that it can play a number of possible roles in the replication of the mitochondrial genome and the maintenance of its telomeres. PMID:26743001

  12. Yeast gene CMR1/YDL156W is consistently co-expressed with genes participating in DNA-metabolic processes in a variety of stringent clustering experiments

    PubMed Central

    Abu-Jamous, Basel; Fa, Rui; Roberts, David J.; Nandi, Asoke K.

    2013-01-01

    The binarization of consensus partition matrices (Bi-CoPaM) method has, among its unique features, the ability to perform ensemble clustering over the same set of genes from multiple microarray datasets by using various clustering methods in order to generate tunable tight clusters. Therefore, we have used the Bi-CoPaM method to the most synchronized 500 cell-cycle-regulated yeast genes from different microarray datasets to produce four tight, specific and exclusive clusters of co-expressed genes. We found 19 genes formed the tightest of the four clusters and this included the gene CMR1/YDL156W, which was an uncharacterized gene at the time of our investigations. Two very recent proteomic and biochemical studies have independently revealed many facets of CMR1 protein, although the precise functions of the protein remain to be elucidated. Our computational results complement these biological results and add more evidence to their recent findings of CMR1 as potentially participating in many of the DNA-metabolism processes such as replication, repair and transcription. Interestingly, our results demonstrate the close co-expressions of CMR1 and the replication protein A (RPA), the cohesion complex and the DNA polymerases α, δ and ɛ, as well as suggest functional relationships between CMR1 and the respective proteins. In addition, the analysis provides further substantial evidence that the expression of the CMR1 gene could be regulated by the MBF complex. In summary, the application of a novel analytic technique in large biological datasets has provided supporting evidence for a gene of previously unknown function, further hypotheses to test, and a more general demonstration of the value of sophisticated methods to explore new large datasets now so readily generated in biological experiments. PMID:23349438

  13. Improved production of fatty acids by Saccharomyces cerevisiae through screening a cDNA library from the oleaginous yeast Yarrowia lipolytica.

    PubMed

    Shi, Shuobo; Ji, Haichuan; Siewers, Verena; Nielsen, Jens

    2016-02-01

    Biological production of fatty acid (FA)-derived products has gained increasing attention to replace petroleum-based fuels and chemicals. FA biosynthesis is highly regulated, and usually it is challenging to design rational engineering strategies. In addition, the conventional 'one sample at a time' method for lipid determination is time consuming and laborious, and it is difficult to screen large numbers of samples. Here, a method for detecting free FAs in viable cells using Nile red staining was developed for use in large-scale screening. Following optimization of the method, it was used for screening a cDNA library from the oleaginous yeast Yarrowia lipolytica for identification of genes/enzymes that were able to enhance free FA accumulation in Saccharomyces cerevisiae. Several novel enzymes resulting in increasing FA accumulation were discovered. These targets include a GPI anchor protein, malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, FA hydroxylase, farnesyltransferase, anoctamin, dihydrolipoamide dehydrogenase and phosphatidylethanolamine-binding protein. The best enzyme resulted in a 2.5-fold improvement in production of free FAs. Our findings not only provide a novel method for high-throughput evaluation of the content of free FAs, but also give new insight into how enzymes from Y. lipolytica may increase the production of fatty acids in S. cerevisiae. PMID:26658002

  14. Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase [alpha

    SciTech Connect

    Sun, Jia; Yang, Yuting; Wan, Ke; Mao, Ninghui; Yu, Tai-Yuan; Lin, Yi-Chien; DeZwaan, Diane C.; Freeman, Brian C.; Lin, Jing-Jer; Lue, Neal F.; Lei, Ming

    2011-08-24

    Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, our structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase {alpha} (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.

  15. Phylogenetic position of the yeast-like symbiotes of Tagosodes orizicolus (Homoptera: Delphacidae) based on 18S ribosomal DNA partial sequences.

    PubMed

    Xet-Mull, Ana M; Quesada, Tania; Espinoza, Ana M

    2004-09-01

    Tagosodes orizicolus Muir (Homoptera: Delphacidae), the endemic delphacid species of tropical America carries yeast-like symbiotes (YLS) in the abdominal fat bodies and the ovarial tissues, like other rice planthoppers of Asia. These YLS are obligate symbiotes, which are transmitted transovarially, and maintain a mutualistic relationship with the insect host. This characteristic has made in vitro culture and classification of YLS rather difficult using conventional methods. Nevertheless, microorganisms of similar characteristics have been successfully classified by using molecular taxonomy. In the present work, the YLS of Tagosodes orizicolus (YLSTo) were purified on Percoll gradients, and specific segments of 18S rDNA were amplified by PCR, cloned and sequenced. Sequences were aligned by means of the CLUSTAL V (DNASTAR) program; phylogenetic trees were constructed with the Phylogeny Inference Package (PHYLIP), showing that YLSTo belong to the fungi class Pyrenomycetes, phylum Ascomycota. Similarities between 98% and 100% were observed among YLS of the rice delphacids Tagosodes orizicolus, Nilaparvata lugens, Laodelphax striatellus and Sogatella fur cifera, and between 89.8% and 90.8% when comparing the above to YLS of the aphid Hamiltonaphis styraci. These comparisons revealed that delphacid YLS are a highly conserved monophyletic group within the Pyrenomycetes and are closely related to Hypomyces chrysospermus. PMID:17361570

  16. Brief Report: Isogenic Induced Pluripotent Stem Cell Lines From an Adult With Mosaic Down Syndrome Model Accelerated Neuronal Ageing and Neurodegeneration

    PubMed Central

    Murray, Aoife; Letourneau, Audrey; Canzonetta, Claudia; Stathaki, Elisavet; Gimelli, Stefania; Sloan‐Bena, Frederique; Abrehart, Robert; Goh, Pollyanna; Lim, Shuhui; Baldo, Chiara; Dagna‐Bricarelli, Franca; Hannan, Saad; Mortensen, Martin; Ballard, David; Syndercombe Court, Denise; Fusaki, Noemi; Hasegawa, Mamoru; Smart, Trevor G.; Bishop, Cleo; Antonarakis, Stylianos E.

    2015-01-01

    Abstract Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well‐controlled cell model systems. We have developed a first nonintegration‐reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high‐resolution whole genome CGH‐array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high‐content microscopic analysis. Early differentiation shows an imbalance of the lineage‐specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC‐derived neurons show increased production of amyloid peptide‐containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21‐derived neurons show significantly higher number of DNA double‐strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21. Stem Cells 2015;33:2077–2084 PMID:25694335

  17. Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro.

    PubMed Central

    Arndt, K M; Ricupero, S L; Eisenmann, D M; Winston, F

    1992-01-01

    A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition. Images PMID:1569955

  18. The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase.

    PubMed Central

    Gangloff, S; McDonald, J P; Bendixen, C; Arthur, L; Rothstein, R

    1994-01-01

    We have previously shown that cells mutant for TOP3, a gene encoding a prokaryotic-like type I topoisomerase in Saccharomyces cerevisiae, display a pleiotropic phenotype including slow growth and genome instability. We identified a mutation, sgs1 (slow growth suppressor), that suppresses both the growth defect and the increased genomic instability of top3 mutants. Here we report the independent isolation of the SGS1 gene in a screen for proteins that interact with Top3. DNA sequence analysis reveals that the putative Sgs1 protein is highly homologous to the helicase encoded by the Escherichia coli recQ gene. These results imply that Sgs1 creates a deleterious topological substrate that Top3 preferentially resolves. The interaction of the Sgs1 helicase homolog and the Top3 topoisomerase is reminiscent of the recently described structure of reverse gyrase from Sulfolobus acidocaldarius, in which a type I DNA topoisomerase and a helicase-like domain are fused in a single polypeptide. Images PMID:7969174

  19. Substitution of conserved residues within the active site alters the cleavage religation equilibrium of DNA topoisomerase I.

    PubMed

    Colley, William C; van der Merwe, Marie; Vance, John R; Burgin, Alex B; Bjornsti, Mary-Ann

    2004-12-24

    Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT). Mutation of conserved residues in close proximity to the active site tyrosine (Tyr(727) of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality. Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates. Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp.DNA complexes result from elevated DNA binding and cleavage. We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants. Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality. Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype. The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. Substitution of Asn(726) with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp. In contrast, replacing the amide side chain of Asn(726) with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding. PMID:15489506

  20. Counting Yeast.

    ERIC Educational Resources Information Center

    Bealer, Jonathan; Welton, Briana

    1998-01-01

    Describes changes to a traditional study of population in yeast colonies. Changes to the procedures include: (1) only one culture per student team; (2) cultures are inoculated only once; and (3) the same tube is sampled daily. (DDR)

  1. Epidemiologic Study of Malassezia Yeasts in Patients with Malassezia Folliculitis by 26S rDNA PCR-RFLP Analysis

    PubMed Central

    Ko, Jong Hyun; Choe, Yong Beom; Ahn, Kyu Joong

    2011-01-01

    Background So far, studies on the inter-relationship between Malassezia and Malassezia folliculitis have been rather scarce. Objective We sought to analyze the differences in body sites, gender and age groups, and to determine whether there is a relationship between certain types of Malassezia species and Malassezia folliculitis. Methods Specimens were taken from the forehead, cheek and chest of 60 patients with Malassezia folliculitis and from the normal skin of 60 age- and gender-matched healthy controls by 26S rDNA PCR-RFLP. Results M. restricta was dominant in the patients with Malassezia folliculitis (20.6%), while M. globosa was the most common species (26.7%) in the controls. The rate of identification was the highest in the teens for the patient group, whereas it was the highest in the thirties for the control group. M. globosa was the most predominant species on the chest with 13 cases (21.7%), and M. restricta was the most commonly identified species, with 17 (28.3%) and 12 (20%) cases on the forehead and cheek, respectively, for the patient group. Conclusion Statistically significant differences were observed between the patient and control groups for the people in their teens and twenties, and in terms of the body site, on the forehead only. PMID:21747616

  2. Asynchronous spore germination in isogenic natural isolates of Saccharomyces paradoxus.

    PubMed

    Stelkens, Rike B; Miller, Eric L; Greig, Duncan

    2016-05-01

    Spores from wild yeast isolates often show great variation in the size of colonies they produce, for largely unknown reasons. Here we measure the colonies produced from single spores from six different wild Saccharomyces paradoxus strains. We found remarkable variation in spore colony sizes, even among spores that were genetically identical. Different strains had different amounts of variation in spore colony sizes, and variation was not affected by the number of preceding meioses, or by spore maturation time. We used time-lapse photography to show that wild strains also have high variation in spore germination timing, providing a likely mechanism for the variation in spore colony sizes. When some spores from a laboratory strain make small colonies, or no colonies, it usually indicates a genetic or meiotic fault. Here, we demonstrate that in wild strains spore colony size variation is normal. We discuss and assess potential adaptive and non-adaptive explanations for this variation. PMID:26880797

  3. An AIF orthologue regulates apoptosis in yeast

    PubMed Central

    Wissing, Silke; Ludovico, Paula; Herker, Eva; Büttner, Sabrina; Engelhardt, Silvia M.; Decker, Thorsten; Link, Alexander; Proksch, Astrid; Rodrigues, Fernando; Corte-Real, Manuela; Fröhlich, Kai-Uwe; Manns, Joachim; Candé, Céline; Sigrist, Stephan J.; Kroemer, Guido; Madeo, Frank

    2004-01-01

    Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1). PMID:15381687

  4. Evolution of p53 transactivation specificity through the lens of a yeast-based functional assay.

    PubMed

    Lion, Mattia; Raimondi, Ivan; Donati, Stefano; Jousson, Olivier; Ciribilli, Yari; Inga, Alberto

    2015-01-01

    Co-evolution of transcription factors (TFs) with their respective cis-regulatory network enhances functional diversity in the course of evolution. We present a new approach to investigate transactivation capacity of sequence-specific TFs in evolutionary studies. Saccharomyces cerevisiae was used as an in vivo test tube and p53 proteins derived from human and five commonly used animal models were chosen as proof of concept. p53 is a highly conserved master regulator of environmental stress responses. Previous reports indicated conserved p53 DNA binding specificity in vitro, even for evolutionary distant species. We used isogenic yeast strains where p53-dependent transactivation was measured towards chromosomally integrated p53 response elements (REs). Ten REs were chosen to sample a wide range of DNA binding affinity and transactivation capacity for human p53 and proteins were expressed at two levels using an inducible expression system. We showed that the assay is amenable to study thermo-sensitivity of frog p53, and that chimeric constructs containing an ectopic transactivation domain could be rapidly developed to enhance the activity of proteins, such as fruit fly p53, that are poorly effective in engaging the yeast transcriptional machinery. Changes in the profile of relative transactivation towards the ten REs were measured for each p53 protein and compared to the profile obtained with human p53. These results, which are largely independent from relative p53 protein levels, revealed widespread evolutionary divergence of p53 transactivation specificity, even between human and mouse p53. Fruit fly and human p53 exhibited the largest discrimination among REs while zebrafish p53 was the least selective. PMID:25668429

  5. Suppressive-subtractive cDNA analysis indicates that elevated yeast antioxidant gene expression is associated with increased stress tolerance and biocontrol efficacy in the antagonistic yeast, Candida Oleophila

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several yeast species have been used as biocontrol agents against postharvest diseases of fruits and vegetables. The objectives of the current study are to develop a better understanding of the role of reactive oxygen species (ROS) in relation to viability and biocontrol activity of Candida oleophi...

  6. Yeast Identification Algorithm Based on Use of the Vitek MS System Selectively Supplemented with Ribosomal DNA Sequencing: Proposal of a Reference Assay for Invasive Fungal Surveillance Programs in China

    PubMed Central

    Zhang, Li; Xiao, Meng; Wang, He; Gao, Ran; Fan, Xin; Brown, Mitchell; Gray, Timothy J.; Kong, Fanrong

    2014-01-01

    Sequence analysis of the internal transcribed spacer (ITS) region was employed as the gold standard method for yeast identification in the China Hospital Invasive Fungal Surveillance Net (CHIF-NET). It has subsequently been found that matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is potentially a more practical approach for this purpose. In the present study, the performance of the Vitek MS v2.0 system for the identification of yeast isolates collected from patients with invasive fungal infections in the 2011 CHIF-NET was evaluated. A total of 1,243 isolates representing 31 yeast species were analyzed, and the identification results by the Vitek MS v2.0 system were compared to those obtained by ITS sequence analysis. By the Vitek MS v2.0 system, 96.7% (n = 1,202) of the isolates were correctly assigned to the species level and 0.2% (n = 2) of the isolates were identified to the genus level, while 2.4% (n = 30) and 0.7% (n = 9) of the isolates were unidentified and misidentified, respectively. After retesting of the unidentified and misidentified strains, 97.3% (n = 1,209) of the isolates were correctly identified to the species level. Based on these results, a testing algorithm that combines the use of the Vitek MS system with selected supplementary ribosomal DNA (rDNA) sequencing was developed and validated for yeast identification purposes. By employing this algorithm, 99.7% (1,240/1,243) of the study isolates were accurately identified with the exception of two isolates of Candida fermentati and one isolate of Cryptococcus gattii. In conclusion, the proposed identification algorithm could be practically implemented in strategic programs of fungal infection surveillance. PMID:24478490

  7. Lager Yeast Comes of Age

    PubMed Central

    2014-01-01

    Alcoholic fermentations have accompanied human civilizations throughout our history. Lager yeasts have a several-century-long tradition of providing fresh beer with clean taste. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. We summarize the initial findings on this hybrid nature, the genomics/transcriptomics of lager yeasts, and established targets of strain improvements. Next-generation sequencing has provided fast access to yeast genomes. Its use in population genomics has uncovered many more hybridization events within Saccharomyces species, so that lager yeast hybrids are no longer the exception from the rule. These findings have led us to propose network evolution within Saccharomyces species. This “web of life” recognizes the ability of closely related species to exchange DNA and thus drain from a combined gene pool rather than be limited to a gene pool restricted by speciation. Within the domesticated lager yeasts, two groups, the Saaz and Frohberg groups, can be distinguished based on fermentation characteristics. Recent evidence suggests that these groups share an evolutionary history. We thus propose to refer to the Saaz group as Saccharomyces carlsbergensis and to the Frohberg group as Saccharomyces pastorianus based on their distinct genomes. New insight into the hybrid nature of lager yeast will provide novel directions for future strain improvement. PMID:25084862

  8. Yeast Droplets

    NASA Astrophysics Data System (ADS)

    Nguyen, Baochi; Upadhyaya, Arpita; van Oudenaarden, Alexander; Brenner, Michael

    2002-11-01

    It is well known that the Young's law and surface tension govern the shape of liquid droplets on solid surfaces. Here we address through experiments and theory the shape of growing aggregates of yeast on agar substrates, and assess whether these ideas still hold. Experiments are carried out on Baker's yeast, with different levels of expressions of an adhesive protein governing cell-cell and cell-substrate adhesion. Changing either the agar concentration or the expression of this protein modifies the local contact angle of a yeast droplet. When the colony is small, the shape is a spherical cap with the contact angle obeying Young's law. However, above a critical volume this structure is unstable, and the droplet becomes nonspherical. We present a theoretical model where this instability is caused by bulk elastic effects. The model predicts that the transition depends on both volume and contact angle, in a manner quantitatively consistent with our experiments.

  9. Genotoxicity of Cytolethal Distending Toxin (CDT) on Isogenic Human Colorectal Cell Lines: Potential Promoting Effects for Colorectal Carcinogenesis

    PubMed Central

    Graillot, Vanessa; Dormoy, Inge; Dupuy, Jacques; Shay, Jerry W.; Huc, Laurence; Mirey, Gladys; Vignard, Julien

    2016-01-01

    The composition of the human microbiota influences tumorigenesis, notably in colorectal cancer (CRC). Pathogenic Escherichia coli possesses a variety of virulent factors, among them the Cytolethal Distending Toxin (CDT). CDT displays dual DNase and phosphatase activities and induces DNA double strand breaks, cell cycle arrest and apoptosis in a broad range of mammalian cells. As CDT could promote malignant transformation, we investigated the cellular outcomes induced by acute and chronic exposures to E. coli CDT in normal human colon epithelial cells (HCECs). Moreover, we conducted a comparative study between isogenic derivatives cell lines of the normal HCECs in order to mimic the mutation of three major genes found in CRC genetic models: APC, KRAS, and TP53. Our results demonstrate that APC and p53 deficient cells showed impaired DNA damage response after CDT exposure, whereas HCECs expressing oncogenic KRASV12 were more resistant to CDT. Compared to normal HCECs, the precancerous derivatives exhibit hallmarks of malignant transformation after a chronic exposure to CDT. HCECs defective in APC and p53 showed enhanced anchorage independent growth and genetic instability, assessed by the micronucleus formation assay. In contrast, the ability to grow independently of anchorage was not impacted by CDT chronic exposure in KRASV12 HCECs, but micronucleus formation is dramatically increased. Thus, CDT does not initiate CRC by itself, but may have promoting effects in premalignant HCECs, involving different mechanisms in function of the genetic alterations associated to CRC. PMID:27047802

  10. Joint Analysis of Near Isogenic and Recombinant Inbred Line Populations Yields Precise Positional Estimates for QTL

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Near isogenic lines (NILs) are typically constructed to fine-map quantitative trait loci (QTL). The data generated for the initial QTL mapping are usually ignored for fine-mapping purposes. However, combining already-available data from initial recombinant inbred line (RIL) studies with new data fro...