Dong-Uk Kim

Korea Research Institute of Bioscience and Biotechnology KRIBB, Daiden, Daejeon, South Korea

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Publications (36)231.66 Total impact


  • No preview · Article · Feb 2016 · The Journal of Microbiology
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    ABSTRACT: Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that an rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs.
    No preview · Article · Nov 2015 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Genome-wide targeted gene deletion, a systematic method to study gene function by replacing target genes with deletion cassettes, using serial-PCR or block-PCR requires elaborate skill. We developed a novel gene-synthesis method to systematically prepare deletion cassettes on a 96-well basis in fission yeast. We designed the 2129-bp deletion cassette as three modules: a central 1397-bp KanMX4 selection marker module and two flanking 366-bp gene-specific artificial linker modules. The central KanMX4 module can be used in multiple deletion cassettes in combination with different sets of flanking modules. The deletion cassettes consisted of 147 oligonucleotides (93 for the central module+25 for each of the flanking modules+4 for the joints) and the oligonucleotides were designed as ~29mers using an in-house program. Oligonucleotides were synthesized on a 96-well basis and ligated into deletion cassettes without gaps by ligase chain reaction, which was followed by two rounds of nested PCR to amplify trace amounts of the ligated cassettes. After the artificial linkers were removed from the deletion cassettes, the cassettes were transformed into wild-type diploid fission yeast strain SP286. We validated the transformed colonies via check PCR and subjected them to tetrad analysis to confirm functional integrity. Using this method, we systematically deleted 563 genes in the fission yeast Schizosaccharomyces pombe with a >90% success rate and a point-mutation rate of ~0.4 mutations per kb. Our method can be used to create systematic gene deletions in a variety of yeasts especially when it included a bar-code system for parallel analyses.
    No preview · Article · Aug 2014 · Journal of Microbiological Methods
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    ABSTRACT: Vorinostat (VOR) has been reported to enhance the cytotoxic effects of doxorubicin (DOX) with fewer side effects because of the lower DOX dosage in breast cancer cells. In this study, we investigated the novel mechanism underlying the synergistic cytotoxic effects of VOR and DOX co-treatment in cervical cancer cells HeLa, CaSki and SiHa cells. Co-treatment with VOR and DOX at marginal doses led to the induction of apoptosis through caspase-3 activation, poly (ADP-ribose) polymerase cleavage and DNA micronuclei. Notably, the synergistic growth inhibition induced by the co-treatment was attributed to the upregulation of the pro-apoptotic protein Bad, as the silencing of Bad expression using small interfering RNA (siRNA) abolished the phenomenon. As siRNA against p53 did not result in an increase in acetylated p53 and the consequent upregulation of Bad, the observed Bad upregulation was mediated by acetylated p53. Moreover, a chromatin immunoprecipitation analysis showed that the co-treatment of HeLa cells with VOR and DOX increased the recruitment of acetylated p53 to the bad promoter, with consequent bad transactivation. Conversely, C33A cervical cancer cells containing mutant p53 co-treated with VOR and DOX did not exhibit Bad upregulation, acetylated p53 induction or consequent synergistic growth inhibition. Together, the synergistic growth inhibition of cervical cancer cell lines induced by co-treatment with VOR and DOX can be attributed to the upregulation of Bad, which is induced by acetylated p53. These results show for the first time that the acetylation of p53, rather than histones, is a mechanism for the synergistic growth inhibition induced by VOR and DOX co-treatments.
    Preview · Article · Feb 2014
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    ABSTRACT: Genome-wide chemical genetic profiles in S. cerevisiae since the budding yeast deletion library construction have been successfully used to reveal unknown mode-of-actions of drugs. Here, we introduce comparative approach to infer drug target proteins more accurately using two compendiums of chemical-genetic profiles from the budding yeast S. cerevisiae and the fission yeast S. pombe. For the first time, we established DNA-chip based growth defect measurement of genome-wide deletion strains of S. pombe, and then applied 47 drugs to the pooled heterozygous deletion strains to generate chemical-genetic profiles in S. pombe. In our approach, putative drug targets were inferred from strains hypersensitive to given drugs by analyzing S. pombe and S. cerevisiae compendiums. Notably, many evidences in the literature revealed that the inferred target genes of fungicide and bactericide identified by such comparative approach are in fact the direct targets. Furthermore, by filtering out the genes with no essentiality, the multi-drug sensitivity genes, and the genes with less eukaryotic conservation, we created a set of drug target gene candidates that are expected to be directly affected by a given drug in human cells. Our study demonstrated that it is highly beneficial to construct the multiple compendiums of chemical genetic profiles using many different species. The fission yeast chemical-genetic compendium is available at http://pombe.kaist.ac.kr/compendium.
    No preview · Article · Jun 2013 · Biochemical and Biophysical Research Communications
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    ABSTRACT: To identify near complete sets of genes required for the cell cycle and cell shape, we have visually screened a genome-wide gene deletion library of 4843 fission yeast deletion mutants (95.7% of total protein encoding genes) for their effects on these processes. A total of 513 genes have been identified as being required for cell cycle progression, 276 of which have not been previously described as cell cycle genes. Deletions of a further 333 genes lead to specific alterations in cell shape and another 524 genes result in generally misshapen cells. Here, we provide the first eukaryotic resource of gene deletions, which describes a near genome-wide set of genes required for the cell cycle and cell shape.
    Full-text · Article · May 2013 · Open Biology
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    Dataset: Figure S3
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    ABSTRACT: Drug sensitivity of S. cerevisiae disomes. (A) Sensitivity of S. cerevisiae disome I (Dis1), II (Dis2), or XIII (Dis13) with indicated CCR4-NOT mutations to CPT. YPD plates with the indicated concentration of CPT were incubated at 30°C for the indicated periods. As a control, haploid strains were also tested. (B) HU-sensitivity of Dis1. (EPS)
    Preview · Dataset · Jun 2012
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    Dataset: Figure S2
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    ABSTRACT: Temperature effect on haploid strains with not3 and not2 deletion mutations. Serial dilutions were spotted as in Figure 3 and incubated at 30°C or at 36°C for 3 d. (EPS)
    Preview · Dataset · Jun 2012
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    Dataset: Table S1
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    ABSTRACT: Effect of the deletion mutant on aneuploid cells. The triploid meiosis shows the S/L ratio (see text for details). The gtub mad2 column indicates whether the gene mutation had a synergistic toxic effect on the gtb1 mad2 double mutant. n: no or little effect; y: appreciable effect. When applicable, the second result was obtained using a different assessment method (see text). The diploid column indicates diploid stability. “1” indicates that this diploid makes a small and deep red colony on the Phloxine B-plate; “2” indicates that the colony size is heterogeneous, especially in the diploid colony; “3” indicates that the haploid makes a small colony; “4” indicates that the colony size is heterogeneous in the haploid colony; “5“ indicates the colony size and color are heterogeneous in the diploid colony; and “6” indicates that the color varies in the diploid colony. ± and −/+ indicate weak and weaker phenotypes, respectively. Cells that have no value indicate the diploid is stable. For those mutants, only the gtub mad2 test was performed, and the diploid stability test was not. The last column shows the instability of the Ch16 minichromosome. ++: highly unstable (∼50% colonies were Ade−); +: unstable (about 10%); ±: mildly unstable (1–2%); −/+: slightly unstable (less than 1%); and n: stable (no Ade− found). (DOC)
    Preview · Dataset · Jun 2012
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    Dataset: Table S2
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    ABSTRACT: Microarray analysis of gene expression in the CCR4-NOT mutants. Genes whose expression changed by at least 1.5-fold in the CCR4-NOT mutants (not3, not2, and caf4). In the two columns next to the fold-change column in each mutant section, a: gene expression was increased at least 1.5-fold in the indicated mutant; b: gene expression decreased at least 1.5-fold; c: gene expression was changed but in the opposite direction. *Genes underexpressed in not3 and/or not2 mapped within a region near the left terminus of chromosome 2; **mapped near the right terminus (see text for details). (XLSX)
    Preview · Dataset · Jun 2012
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    Dataset: Table S3
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    ABSTRACT: S. cerevisiae disomes and control haploid strains used in the present study. (XLSX)
    Preview · Dataset · Jun 2012
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    Dataset: Figure S1
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    ABSTRACT: Synergistic effects of the indicated mutants on the gtb1 mad2 double mutant. See Figure 2 legend for details. (TIF)
    Preview · Dataset · Jun 2012
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    ABSTRACT: To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, 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 Saccharomyces cerevisiae 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.
    Full-text · Article · Jun 2012 · PLoS Genetics
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    ABSTRACT: Type 1 phosphatase (PP1) antagonizes Aurora B kinase to stabilize kinetochore-microtubule attachments and to silence the spindle checkpoint. We screened for factors that exacerbate the growth defect of Δdis2 cells, which lack one of two catalytic subunits of PP1 in fission yeast, and identified Nsk1, a novel protein required for accurate chromosome segregation. During interphase, Nsk1 resides in the nucleolus but spreads throughout the nucleoplasm as cells enter mitosis. Following dephosphorylation by Clp1 (Cdc14-like) phosphatase and at least one other phosphatase, Nsk1 localizes to the interface between kinetochores and the inner face of the spindle pole body during anaphase. In the absence of Nsk1, some kinetochores become detached from spindle poles during anaphase B. If this occurs late in anaphase B, then the sister chromatids of unclustered kinetochores segregate to the correct daughter cell. These unclustered kinetochores are efficiently captured, retrieved, bioriented, and segregated during the following mitosis, as long as Dis2 is present. However, if kinetochores are detached from a spindle pole early in anaphase B, then these sister chromatids become missegregated. These data suggest Nsk1 ensures accurate chromosome segregation by promoting the tethering of kinetochores to spindle poles during anaphase B.
    Full-text · Article · Sep 2011 · Molecular biology of the cell
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    ABSTRACT: 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 defective for SREBP cleavage, dsc1-4, 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. dsc 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.
    Full-text · Article · Apr 2011 · Molecular cell
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    ABSTRACT: 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(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² 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(Cdt)² ubiquitin ligase-dependent Spd1 degradation and RNR activation promotes postsynaptic ssDNA gap filling during HR repair.
    Full-text · Article · Dec 2010 · Genes & Development
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    ABSTRACT: The fission yeast Schizosaccharomyces pombe is a model organism used widely to study various aspects of eukaryotic biology. A collection of heterozygous diploid strains containing individual deletions in nearly all S. pombe genes has been created using a PCR based strategy. However, deletion of some genes has not been possible using this methodology. Here we use an efficient knockout strategy based on plasmids that contain large regions homologous to the target gene to delete an additional 29 genes. The collection of deletion mutants now covers 99% of the fission yeast open reading frames.
    Full-text · Article · Jun 2010 · Cell cycle (Georgetown, Tex.)
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    ABSTRACT: 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.
    Full-text · Article · Jun 2010 · Nature Biotechnology
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    ABSTRACT: In fission yeast, RNAi directs heterochromatin formation at centromeres, telomeres, and the mating type locus. Noncoding RNAs transcribed from repeat elements generate siRNAs that are incorporated into the Argonaute-containing RITS complex and direct it to nascent homologous transcripts. This leads to recruitment of the CLRC complex, including the histone methyltransferase Clr4, promoting H3K9 methylation and heterochromatin formation. A key question is what mediates the recruitment of Clr4/CLRC to transcript-bound RITS. We have identified a LIM domain protein, Stc1, that is required for centromeric heterochromatin integrity. Our analyses show that Stc1 is specifically required to establish H3K9 methylation via RNAi, and interacts both with the RNAi effector Ago1, and with the chromatin-modifying CLRC complex. Moreover, tethering Stc1 to a euchromatic locus is sufficient to induce silencing and heterochromatin formation independently of RNAi. We conclude that Stc1 associates with RITS on centromeric transcripts and recruits CLRC, thereby coupling RNAi to chromatin modification.
    Full-text · Article · Mar 2010 · Cell
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    ABSTRACT: 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, Candida albicans and Saccharomyces cerevisiae. Recently, the much more distant fission yeast Schizosaccharomyces pombe 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 S. pombe, on rich media. Screening of an S. pombe haploid deletion library identified 12 genes required for mycelial development which encode potential transcription factors, orthologues of S. cerevisiae 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.
    Preview · Article · Jul 2009 · Eukaryotic Cell