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Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe
Kim, Dong-Uk,Hayles, Jacqueline,Kim, Dongsup,Wood, Valerie,Park, Han-Oh,Won, Misun,Yoo, Hyang-Sook,Duhig, Trevor,Nam, Miyoung,Palmer, Georgia,Han, Sangjo,Jeffery, Linda,Baek, Seung-Tae,Lee, Hyemi,Shim Nature Publishing Group 2010 Nature biotechnology Vol.28 No.6
We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome providing a tool for studying eukaryotic biology. Comprehensive gene dispensability comparisons with budding yeast—the only other eukaryote for which a comprehensive knockout library exists—revealed that 83% of single-copy orthologs in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than nonessential genes to be present in a single copy, to be broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth.
Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe
Kim, Dong-Uk,Hayles, Jacqueline,Kim, Dongsup,Wood, Valerie,Park, Han-Oh,Won, Misun,Yoo, Hyang-Sook,Duhig, Trevor,Nam, Miyoung,Palmer, Georgia,Han, Sangjo,Jeffery, Linda,Baek, Seung-Tae,Lee, Hyemi,Shim Nature Publishing Group, a division of Macmillan P 2010 Nature biotechnology Vol.28 No.12
Jansson, K.,Warringer, J.,Farewell, A.,Park, H.O.,Hoe, K.L.,Kim, D.U.,Hayles, J.,Sunnerhagen, P. Elsevier 2008 Mutation research Vol.644 No.1
The DNA glycosylase MutY is strongly conserved in evolution, and homologs are found in most eukaryotes and prokaryotes examined. This protein is implicated in repair of oxidative DNA damage, in particular adenine mispaired opposite 7,8-dihydro-8-oxoguanine. Previous investigations in Escherichia coli, fission yeast, and mammalian cells show an association of mutations in MutY homologs with a mutator phenotype and carcinogenesis. Eukaryotic MutY homologs physically associate with several proteins with a role in replication, DNA repair, and checkpoint signaling, specifically the trimeric 9-1-1 complex. In a genetic investigation of the fission yeast MutY homolog, myh1<SUP>+</SUP>, we show that the myh1 mutation confers a moderately increased UV sensitivity alone and in combination with mutations in several DNA repair genes. The myh1 rad1, and to a lesser degree myh1 rad9, double mutants display a synthetic interaction resulting in enhanced sensitivity to DNA damaging agents and hydroxyurea. UV irradiation of myh1 rad1 double mutants results in severe chromosome segregation defects and visible DNA fragmentation, and a failure to activate the checkpoint. Additionally, myh1 rad1 double mutants exhibit morphological defects in the absence of DNA damaging agents. We also found a moderate suppression of the slow growth and UV sensitivity of rhp51 mutants by the myh1 mutation. Our results implicate fission yeast Myh1 in repair of a wider range of DNA damage than previously thought, and functionally link it to the checkpoint pathway.
The CCR4-NOT Complex Is Implicated in the Viability of Aneuploid Yeasts
Tange, Yoshie,Kurabayashi, Atsushi,Goto, Bunshiro,Hoe, Kwang-Lae,Kim, Dong-Uk,Park, Han-Oh,Hayles, Jacqueline,Chikashige, Yuji,Tsutumi, Chihiro,Hiraoka, Yasushi,Yamao, Fumiaki,Nurse, Paul,Niwa, Osami Public Library of Science 2012 PLoS genetics Vol.8 No.6
<P>To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast <I>Schizosaccharomyces pombe</I>, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast <I>Saccharomyces cerevisiae</I> also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability.</P><P><B>Author Summary</B></P> <P>Aneuploidy is a major cause of abortive development and is implicated in tumorigenesis in humans. Recent studies revealed that the increased need for protein degradation might account for the detrimental effects of aneuploidy on a cell. Here, we investigated the genetic systems responsible for aneuploid viability. Using a collection of gene deletions in fission yeast, we isolated mutants that affect aneuploid viability. We found that an evolutionarily conserved transcription regulator, the CCR4-NOT complex, and its related factors are required for aneuploid viability, suggesting that regulation of mRNA turnover is required to tolerate aneuploidy. In addition, homologous recombination repair is important for aneuploid viability.</P>
Functional Genomics of Adhesion, Invasion, and Mycelial Formation in Schizosaccharomyces pombe
Dodgson, James,Avula, Hema,Hoe, Kwang-Lae,Kim, Dong-Uk,Park, Han-Oh,Hayles, Jacqueline,Armstrong, John American Society for Microbiology 2009 EUKARYOTIC CELL Vol.8 No.8
<B>ABSTRACT</B><P>Investigation into the switch between single-celled and filamentous forms of fungi may provide insights into cell polarity, differentiation, and fungal pathogenicity. At the molecular level, much of this investigation has fallen on two closely related budding yeasts, <I>Candida albicans</I> and <I>Saccharomyces cerevisiae</I>. Recently, the much more distant fission yeast <I>Schizosaccharomyces pombe</I> was shown to form invasive filaments after nitrogen limitation (E. Amoah-Buahin, N. Bone, and J. Armstrong, Eukaryot. Cell 4:1287-1297, 2005) and this genetically tractable organism provides an alternative system for the study of dimorphic growth. Here we describe a second mode of mycelial formation of <I>S. pombe</I>, on rich media. Screening of an <I>S. pombe</I> haploid deletion library identified 12 genes required for mycelial development which encode potential transcription factors, orthologues of <I>S. cerevisiae</I> Sec14p and Tlg2p, and the formin For3, among others. These were further grouped into two phenotypic classes representing different stages of the process. We show that galactose-dependent cell adhesion and actin assembly are both required for mycelial formation and mutants lacking a range of genes controlling cell polarity all produce mycelia but with radically altered morphology.</P>
Moss, Jennifer,Tinline-Purvis, Helen,Walker, Carol A,Folkes, Lisa K,Stratford, Michael R,Hayles, Jacqueline,Hoe, Kwang-Lae,Kim, Dong-Uk,Park, Han-Oh,Kearsey, Stephen E,Fleck, Oliver,Holmberg, Christia Cold Spring Harbor Laboratory in association with 2010 Genes & development Vol.24 No.23
<P>Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found the Ddb1-Cul4(Cdt2) ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt2) ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1(+) suppressed the DNA damage sensitivity and the reduced HR efficiency associated with loss of ddb1(+) or cdt2(+). Furthermore, we demonstrate a role for nucleotide synthesis in postsynaptic gap filling of resected ssDNA ends during HR repair. Finally, we define a role for Rad3 (ATR) in nucleotide synthesis and HR through increasing Cdt2 nuclear levels in response to DNA damage. Our findings support a model in which break-induced Rad3 and Ddb1-Cul4(Cdt2) ubiquitin ligase-dependent Spd1 degradation and RNR activation promotes postsynaptic ssDNA gap filling during HR repair.</P>
Yeast SREBP Cleavage Activation Requires the Golgi Dsc E3 Ligase Complex
Stewart, Emerson ,V.,Nwosu, Christine ,C.,Tong, Zongtian,Roguev, Assen,Cummins, Timothy ,D.,Kim, Dong-Uk,Hayles, Jacqueline,Park, Han-Oh,Hoe, Kwang-Lae,Powell, David ,W.,Krogan, Nevan& Elsevier 2011 Molecular cell Vol.42 No.2
<P><B>Summary</B></P><P>Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes <I>d</I>efective for <I>S</I>REBP <I>c</I>leavage, <I>dsc1-4</I>, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2-conjugating enzyme Ubc4, the Dsc1 RING E3 ligase, and the proteasome. <I>dsc</I> mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.</P> <P><B>Graphical Abstract</B></P><P><ce:figure id='dfig1'></ce:figure></P><P><B>Highlights</B></P><P>► Yeast SREBP is proteolytically activated by a different mechanism than mammalian SREBP ► Deletion collection screen identified four <I>dsc</I> genes required for fission yeast SREBP cleavage ► Dsc proteins form a Golgi E3 ligase complex that resembles Hrd1 E3 ligase in ERAD ► Yeast SREBP cleavage requires activities of the ubiquitin-proteasome pathway</P>
A genome-wide screen of genes involved in cadmium tolerance in Schizosaccharomyces pombe.
Kennedy, Patrick J,Vashisht, Ajay A,Hoe, Kwang-Lae,Kim, Dong-Uk,Park, Han-Oh,Hayles, Jacqueline,Russell, Paul Academic Press 2008 TOXICOLOGICAL SCIENCES Vol.106 No.1
<P>Cadmium is a worldwide environmental toxicant responsible for a range of human diseases including cancer. Cellular injury from cadmium is minimized by stress-responsive detoxification mechanisms. We explored the genetic requirements for cadmium tolerance by individually screening mutants from the fission yeast (Schizosaccharomyces pombe) haploid deletion collection for inhibited growth on agar growth media containing cadmium. Cadmium-sensitive mutants were further tested for sensitivity to oxidative stress (hydrogen peroxide) and osmotic stress (potassium chloride). Of 2649 mutants screened, 237 were sensitive to cadmium, of which 168 were cadmium specific. Most were previously unknown to be involved in cadmium tolerance. The 237 genes represent a number of pathways including sulfate assimilation, phytochelatin synthesis and transport, ubiquinone (Coenzyme Q10) biosynthesis, stress signaling, cell wall biosynthesis and cell morphology, gene expression and chromatin remodeling, vacuole function, and intracellular transport of macromolecules. The ubiquinone biosynthesis mutants are acutely sensitive to cadmium but only mildly sensitive to hydrogen peroxide, indicating that Coenzyme Q10 plays a larger role in cadmium tolerance than just as an antioxidant. These and several other mutants turn yellow when exposed to cadmium, suggesting cadmium sulfide accumulation. This phenotype can potentially be used as a biomarker for cadmium. There is remarkably little overlap with a comparable screen of the Saccharomyces cerevisiae haploid deletion collection, indicating that the two distantly related yeasts utilize significantly different strategies for coping with cadmium stress. These strategies and their relation to cadmium detoxification in humans are discussed.</P>