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Author McPheeters, D.S.; Wise, J.A. file  url
openurl 
  Title Measurement of in vivo RNA synthesis rates Type Journal Article
  Year (down) 2013 Publication Methods in Enzymology Abbreviated Journal Methods Enzymol  
  Volume 530 Issue Pages 117-135  
  Keywords Gene Expression Regulation, Fungal; RNA, Fungal/*genetics; Saccharomyces cerevisiae/*genetics; Schizosaccharomyces/*genetics; Transcription, Genetic; Immobilized DNA/RNA; Immobilized probes; In vivo RNA synthesis rates; Labeled RNA; Nascent transcripts  
  Abstract A technique is described to directly measure ongoing transcription from individual genes in permeabilized cells of either the budding yeast Saccharomyces cerevisiae or the fission yeast Schizosaccharomyces pombe. Transcription run-on (TRO) analysis is used to compare the relative rates of synthesis for specific transcripts in cells grown under different environmental conditions or harvested at different stages of development. As the amount of an individual RNA species present at any given time is determined by its net rate of synthesis and degradation, an accurate picture of transcription per se can be obtained only by directly measuring de novo synthesis of RNA (if you are interested in RNA degradation, see Method for measuring mRNA decay rate in Saccharomyces cerevisiae). Most techniques employed to measure changes in the relative levels of individual transcripts present under different conditions, including Northern analysis (see Northern blotting), RT-PCR (see Reverse-transcription PCR (RT-PCR)), nuclease protection assays (see Explanatory Chapter: Nuclease Protection Assays), and genome-wide assays, such as microarray analysis and high throughput RNA sequencing, measure changes in the steady-state level of a transcript, which may or may not reflect the actual changes in transcription of the gene. Recent studies carried out in fission yeast have demonstrated that increases in the steady-state level (accumulation) of many individual mRNAs occur without any significant changes in transcription rates (McPheeters et al., 2009), highlighting the important role of regulated RNA stability in determining gene expression programs (Harigaya et al., 2006).  
  Call Number Serial 1345  
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Author Schenone, M.; Dancik, V.; Wagner, B.K.; Clemons, P.A. file  url
openurl 
  Title Target identification and mechanism of action in chemical biology and drug discovery Type Journal Article
  Year (down) 2013 Publication Nature Chemical Biology Abbreviated Journal Nat Chem Biol  
  Volume 9 Issue 4 Pages 232-240  
  Keywords Animals; Biomarkers, Pharmacological/chemistry/*metabolism; *Drug Discovery; *Drug Evaluation, Preclinical; *High-Throughput Screening Assays; Humans; Isotope Labeling; Mass Spectrometry; Molecular Targeted Therapy; Phenotype; RNA Interference; Reverse Genetics; Saccharomyces cerevisiae/drug effects/genetics/metabolism; Small Molecule Libraries/chemistry/*metabolism/pharmacology; Validation Studies as Topic  
  Abstract Target-identification and mechanism-of-action studies have important roles in small-molecule probe and drug discovery. Biological and technological advances have resulted in the increasing use of cell-based assays to discover new biologically active small molecules. Such studies allow small-molecule action to be tested in a more disease-relevant setting at the outset, but they require follow-up studies to determine the precise protein target or targets responsible for the observed phenotype. Target identification can be approached by direct biochemical methods, genetic interactions or computational inference. In many cases, however, combinations of approaches may be required to fully characterize on-target and off-target effects and to understand mechanisms of small-molecule action.  
  Call Number Serial 1592  
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Author Schmidt, B.H.; Osheroff, N.; Berger, J.M. file  url
openurl 
  Title Structure of a topoisomerase II-DNA-nucleotide complex reveals a new control mechanism for ATPase activity Type Journal Article
  Year (down) 2012 Publication Nature Structural & Molecular Biology Abbreviated Journal Nat Struct Mol Biol  
  Volume 19 Issue 11 Pages 1147-1154  
  Keywords Adenylyl Imidodiphosphate/*chemistry/metabolism; Amino Acid Sequence; Antigens, Neoplasm/*chemistry/metabolism; Chromatography, Gel; Crystallization; DNA/*chemistry/metabolism; DNA Topoisomerases, Type II/*chemistry/metabolism; DNA-Binding Proteins/*chemistry/metabolism; Dimerization; *Models, Molecular; Molecular Sequence Data; Multiprotein Complexes/*chemistry/metabolism; *Protein Conformation; Saccharomyces cerevisiae/*enzymology  
  Abstract Type IIA topoisomerases control DNA supercoiling and disentangle chromosomes through a complex ATP-dependent strand-passage mechanism. Although a general framework exists for type IIA topoisomerase function, the architecture of the full-length enzyme has remained undefined. Here we present the structure of a fully catalytic Saccharomyces cerevisiae topoisomerase II homodimer complexed with DNA and a nonhydrolyzable ATP analog. The enzyme adopts a domain-swapped configuration wherein the ATPase domain of one protomer sits atop the nucleolytic region of its partner subunit. This organization produces an unexpected interaction between bound DNA and a conformational transducing element in the ATPase domain, which we show is critical for both DNA-stimulated ATP hydrolysis and global topoisomerase activity. Our data indicate that the ATPase domains pivot about each other to ensure unidirectional strand passage and that this state senses bound DNA to promote ATP turnover and enzyme reset.  
  Call Number Serial 2189  
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Author Yogev, O.; Yogev, O.; Singer, E.; Shaulian, E.; Goldberg, M.; Fox, T.D.; Pines, O. file  url
openurl 
  Title Fumarase: a mitochondrial metabolic enzyme and a cytosolic/nuclear component of the DNA damage response Type Journal Article
  Year (down) 2010 Publication PLoS Biology Abbreviated Journal PLoS Biol  
  Volume 8 Issue 3 Pages e1000328  
  Keywords Cell Nucleus/*metabolism; Cytosol/*metabolism; *DNA Damage; Fumarate Hydratase/genetics/*metabolism; Fumarates/metabolism; Gene Knockdown Techniques; HeLa Cells; Histones/genetics/metabolism; Humans; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism; Isoenzymes/genetics/*metabolism; Kidney Neoplasms/enzymology/genetics; Leiomyomatosis/enzymology/genetics; Mitochondria/*enzymology; Saccharomyces cerevisiae/enzymology/genetics; Saccharomyces cerevisiae Proteins/genetics/metabolism; Tumor Suppressor Proteins/genetics/metabolism  
  Abstract In eukaryotes, fumarase (FH in human) is a well-known tricarboxylic-acid-cycle enzyme in the mitochondrial matrix. However, conserved from yeast to humans is a cytosolic isoenzyme of fumarase whose function in this compartment remains obscure. A few years ago, FH was surprisingly shown to underlie a tumor susceptibility syndrome, Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). A biallelic inactivation of FH has been detected in almost all HLRCC tumors, and therefore FH was suggested to function as a tumor suppressor. Recently it was suggested that FH inhibition leads to elevated intracellular fumarate, which in turn acts as a competitive inhibitor of HPH (HIF prolyl hydroxylase), thereby causing stabilization of HIF (Hypoxia-inducible factor) by preventing proteasomal degradation. The transcription factor HIF increases the expression of angiogenesis regulated genes, such as VEGF, which can lead to high microvessel density and tumorigenesis. Yet this mechanism does not fully explain the large cytosolic population of fumarase molecules. We constructed a yeast strain in which fumarase is localized exclusively to mitochondria. This led to the discovery that the yeast cytosolic fumarase plays a key role in the protection of cells from DNA damage, particularly from DNA double-strand breaks. We show that the cytosolic fumarase is a member of the DNA damage response that is recruited from the cytosol to the nucleus upon DNA damage induction. This function of fumarase depends on its enzymatic activity, and its absence in cells can be complemented by high concentrations of fumaric acid. Our findings suggest that fumarase and fumaric acid are critical elements of the DNA damage response, which underlies the tumor suppressor role of fumarase in human cells and which is most probably HIF independent. This study shows an exciting crosstalk between primary metabolism and the DNA damage response, thereby providing a scenario for metabolic control of tumor propagation.  
  Call Number Serial 1880  
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Author Mazzoni, C.; Falcone, C. file  url
openurl 
  Title Caspase-dependent apoptosis in yeast Type Journal Article
  Year (down) 2008 Publication Biochimica et Biophysica Acta Abbreviated Journal Biochim Biophys Acta  
  Volume 1783 Issue 7 Pages 1320-1327  
  Keywords Apoptosis--genetics, physiology; Apoptosis Regulatory Proteins--metabolism; Caspases--metabolism; Mitochondria--metabolism; Saccharomyces cerevisiae--genetics, physiology; Saccharomyces cerevisiae Proteins--metabolism; Signal Transduction  
  Abstract Damaging environment, certain intracellular defects or heterologous expression of pro-apoptotic genes induce death in yeast cells exhibiting typical markers of apoptosis. In mammals, apoptosis can be directed by the activation of groups of proteases, called caspases, that cleave specific substrates and trigger cell death. In addition, in plants, fungi, Dictyostelium and metazoa, paracaspases and metacaspases have been identified that share some homologies with caspases but showing different substrate specificity. In the yeast Saccharomyces cerevisiae, a gene (MCA1/YCA1) has been identified coding for a metacaspase involved in the induction of cell death. Metacaspases are not biochemical, but sequence and functional homologes of caspases, as deletion of them rescues entirely different death scenarios. In this review we will summarize the current knowledge in S. cerevisiae on apoptotic processes, induced by internal and external triggers, which are dependent on the metacaspase gene YCA1.  
  Call Number Serial 850  
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Author Bieling, P.; Laan, L.; Schek, H.; Munteanu, E.L.; Sandblad, L.; Dogterom, M.; Brunner, D.; Surrey, T. file  url
openurl 
  Title Reconstitution of a microtubule plus-end tracking system in vitro Type Journal Article
  Year (down) 2007 Publication Nature Abbreviated Journal Nature  
  Volume 450 Issue 7172 Pages 1100-1105  
  Keywords  
  Abstract The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.

Subject Heading: Cell-Free System; Heat-Shock Proteins/metabolism; Intermediate Filament Proteins/metabolism; Microscopy, Fluorescence; Microtubule-Associated Proteins/*metabolism; Microtubules/*chemistry/*metabolism; *Schizosaccharomyces/chemistry/cytology; Schizosaccharomyces pombe Proteins/metabolism
 
  Call Number Serial 2223  
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Author Gupta, M.L.J.; Carvalho, P.; Roof, D.M.; Pellman, D. file  url
openurl 
  Title Plus end-specific depolymerase activity of Kip3, a kinesin-8 protein, explains its role in positioning the yeast mitotic spindle Type Journal Article
  Year (down) 2006 Publication Nature Cell Biology Abbreviated Journal Nat Cell Biol  
  Volume 8 Issue 9 Pages 913-923  
  Keywords  
  Abstract The budding yeast protein Kip3p is a member of the conserved kinesin-8 family of microtubule motors, which are required for microtubule-cortical interactions, normal spindle assembly and kinetochore dynamics. Here, we demonstrate that Kip3p is both a plus end-directed motor and a plus end-specific depolymerase--a unique combination of activities not found in other kinesins. The ATPase activity of Kip3p was activated by both microtubules and unpolymerized tubulin. Furthermore, Kip3p in the ATP-bound state formed a complex with unpolymerized tubulin. Thus, motile kinesin-8s may depolymerize microtubules by a mechanism that is similar to that used by non-motile kinesin-13 proteins. Fluorescent speckle analysis established that, in vivo, Kip3p moved toward and accumulated on the plus ends of growing microtubules, suggesting that motor activity brings Kip3p to its site of action. Globally, and more dramatically on cortical contact, Kip3p promoted catastrophes and pausing, and inhibited microtubule growth. These findings explain the role of Kip3p in positioning the mitotic spindle in budding yeast and potentially other processes controlled by kinesin-8 family members.

Subject headings: Adenosine Triphosphatases/metabolism; Cell Cycle/physiology; Kinesin/*metabolism; Microtubule-Associated Proteins/*physiology; Microtubules/*physiology; Molecular Motor Proteins/*physiology; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins/*physiology; Spindle Apparatus/*physiology; Tubulin/metabolism
 
  Call Number Serial 2212  
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Author Westermann, S.; Wang, H.-W.; Avila-Sakar, A.; Drubin, D.G.; Nogales, E.; Barnes, G. file  url
openurl 
  Title The Dam1 kinetochore ring complex moves processively on depolymerizing microtubule ends Type Journal Article
  Year (down) 2006 Publication Nature Abbreviated Journal Nature  
  Volume 440 Issue 7083 Pages 565-569  
  Keywords  
  Abstract Chromosomes interact through their kinetochores with microtubule plus ends and they are segregated to the spindle poles as the kinetochore microtubules shorten during anaphase A of mitosis. The molecular natures and identities of coupling proteins that allow microtubule depolymerization to pull chromosomes to poles during anaphase have long remained elusive. In budding yeast, the ten-protein Dam1 complex is a critical microtubule-binding component of the kinetochore that oligomerizes into a 50-nm ring around a microtubule in vitro. Here we show, with the use of a real-time, two-colour fluorescence microscopy assay, that the ring complex moves processively for several micrometres at the ends of depolymerizing microtubules without detaching from the lattice. Electron microscopic analysis of 'end-on views' revealed a 16-fold symmetry of the kinetochore rings. This out-of-register arrangement with respect to the 13-fold microtubule symmetry is consistent with a sliding mechanism based on an electrostatically coupled ring-microtubule interface. The Dam1 ring complex is a molecular device that can translate the force generated by microtubule depolymerization into movement along the lattice to facilitate chromosome segregation.

Subject Headings: Cell Cycle Proteins/*physiology; Chromosome Segregation/physiology; Kinetochores/*physiology/ultrastructure; Microscopy, Fluorescence; Microtubule-Associated Proteins/*physiology; Microtubules/*physiology/ultrastructure; Movement; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins/*physiology; Spindle Apparatus/*physiology/ultrastructure
 
  Call Number Serial 2219  
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Author Gupta, M.L.J.; Carvalho, P.; Roof, D.M.; Pellman, D. file  url
openurl 
  Title Plus end-specific depolymerase activity of Kip3, a kinesin-8 protein, explains its role in positioning the yeast mitotic spindle Type Journal Article
  Year (down) 2006 Publication Nature Cell Biology Abbreviated Journal Nat Cell Biol  
  Volume 8 Issue 9 Pages 913-923  
  Keywords  
  Abstract The budding yeast protein Kip3p is a member of the conserved kinesin-8 family of microtubule motors, which are required for microtubule-cortical interactions, normal spindle assembly and kinetochore dynamics. Here, we demonstrate that Kip3p is both a plus end-directed motor and a plus end-specific depolymerase--a unique combination of activities not found in other kinesins. The ATPase activity of Kip3p was activated by both microtubules and unpolymerized tubulin. Furthermore, Kip3p in the ATP-bound state formed a complex with unpolymerized tubulin. Thus, motile kinesin-8s may depolymerize microtubules by a mechanism that is similar to that used by non-motile kinesin-13 proteins. Fluorescent speckle analysis established that, in vivo, Kip3p moved toward and accumulated on the plus ends of growing microtubules, suggesting that motor activity brings Kip3p to its site of action. Globally, and more dramatically on cortical contact, Kip3p promoted catastrophes and pausing, and inhibited microtubule growth. These findings explain the role of Kip3p in positioning the mitotic spindle in budding yeast and potentially other processes controlled by kinesin-8 family members.

Subject Headings: Adenosine Triphosphatases/metabolism; Cell Cycle/physiology; Kinesin/*metabolism; Microtubule-Associated Proteins/*physiology; Microtubules/*physiology; Molecular Motor Proteins/*physiology; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins/*physiology; Spindle Apparatus/*physiology; Tubulin/metabolism
 
  Call Number Serial 2260  
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Author Heinisch, J.J. file  url
doi  openurl
  Title Baker's yeast as a tool for the development of antifungal kinase inhibitors--targeting protein kinase C and the cell integrity pathway Type Journal Article
  Year (down) 2005 Publication Biochimica et Biophysica Acta Abbreviated Journal Biochim Biophys Acta  
  Volume 1754 Issue 1-2 Pages 171-182  
  Keywords Antifungal Agents/*chemistry/metabolism/pharmacology; Cell Cycle/*drug effects; Cell Wall/drug effects/metabolism; Enzyme Inhibitors/*chemistry/metabolism/pharmacology; Humans; MAP Kinase Signaling System/drug effects; Models, Biological; Protein Kinase C/*antagonists & inhibitors/chemistry/drug effects/metabolism; Protein Kinases/genetics/metabolism; Recombinant Fusion Proteins/chemistry/*metabolism; Saccharomyces cerevisiae/chemistry/enzymology/*metabolism; Saccharomyces cerevisiae Proteins/*antagonists & inhibitors/chemistry/drug effects/metabolism  
  Abstract Today, the yeast Saccharomyces cerevisiae is probably the best-studied eukaryotic organism. This review first focuses on the signaling process which is mediated by the unique yeast protein kinase C (Pkc1p) and a downstream mitogen-activated protein kinase (MAPK) cascade. This pathway ensures cellular integrity by sensing cell surface stress and controlling cell wall biosynthesis and progression through the cell cycle. The domain structure of Pkc1p is conserved from yeast to humans. A yeast system for heterologous expression of specific domains in a chimeric yeast/mammalian PKC enzyme (“domain shuffling”) is depicted. It is also proposed how this system could be employed for the study of protein kinase inhibitors in high-throughput screens. Moreover, a reporter assay that allows a quantitative readout of the activity of the cell integrity signaling pathway is introduced. Since a variety of protein kinases take part in the signal transduction, this broadens the range of targets for potential inhibitors.  
  Call Number Serial 554  
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