DOCSLIB.ORG

Ribonuclease T:New Exoribonuclease Possibly Involved in End-Turnover

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Ribonuclease T:New Exoribonuclease Possibly Involved in End-Turnover Proc. Natl. Acad. Sci. USA Vol. 81, pp. 4290-4293, July 1984 Biochemistry Ribonuclease T: New exoribonuclease possibly involved in end-turnover of tRNA (Escherichia coli/3' terminus) MURRAY P. DEUTSCHER, CHRISTOPHER W. MARLOR, AND RICHARD ZANIEWSKI Department of Biochemistry, University of Connecticut Health Center, Farmington, CT 06032 Communicated by M. J. Osborn, March 26, 1984 ABSTRACT Examination of double mutants lacking one provide evidence that it is distinct from the previously de- of the exoribonucleases, RNase II, RNase D, RNase BN, or scribed exoribonucleases of E. coli. RNase R, and also devoid of tRNA nucleotidyltransferase has suggested that none of these RNases participates in the end- MATERIALS AND METHODS turnover of tRNA. This prompted a search for and identifica- Bacterial Strains. All strains used in this study are E. coli tion of a new exoribonuclease, termed RNase T. RNase T K-12 derivatives. The RNase II- strain, C3b; the tempera- could be detected in mutant Escherichia coli strains lacking as ture-sensitive RNase Dts strain, C3c; the RNase BN- strain, many as three of the known exoribonucleases, and it could be CAN; and its parent strain, CA265, have been described separated from each of the four previously described RNases. (10-12). C3 and CAN derivatives containing an rnd deletion, RNase T is optimally active at pH 8-9 and requires a divalent strains C3/5 and CAN/1, were prepared by using Pl-mediat- cation for activity. The enzyme is sensitive to ionic strengths > ed transduction to transfer the deletion from strain RB14 50 mM and is rapidly inactivated by heating at 45°C. Its pre- (13). For this purpose a TnJO transposon was inserted adja- ferred substrate is tRNA-C-C-[14C]A, with much less activity cent to the deletion, and tetracycline-resistant transductants shown against tRNA-C-C. RNase T is an exoribonuclease that were assayed for the absence of RNase D activity. The initiates attack at the 3' hydroxyl terminus of tRNA and re- RNase R- strain, S296-6808, was constructed by reintroduc- leases AMP in a random mode of hydrolysis. The possible in- ing rnb+ into strain S296-680 obtained from David Schles- volvement of RNase T in end-turnover of tRNA and in RNA singer (Washington University), using cotransduction with metabolism in general are discussed. trp+ (6), and assaying for transductants containing RNase II activity. In recent years it has become apparent that cells contain a Strain CAN/20-12 (lacking RNase II, RNase D, and multitude of distinct ribonucleases that participate in the var- RNase BN) and strain CA265/3-1 (lacking RNase II and ious degradative and processing reactions of RNA metabo- RNase D) were constructed by introduction of the rnb and lism (see ref. 1 for examples). Even in the case of exoribonu- rnd mutations into strains CAN and CA265. The strains were cleases, which generally have been considered to be nonspe- first made trp- by transduction of the trp: :TnJO marker from cific degradative enzymes, it is now clear that a variety of strain NK5151 (obtained from Nancy Kleckner, Harvard these activities with different specificities are present in a University). The rnb mutation was introduced by cotrans- single cell. Thus, four exoribonucleases have been identified duction with trp+ using P1 grown on strain C3b to generate previously in Escherichia coli (a fifth one that also may be strains CAN/20 and CA265/3. The rnd deletion was then considered is polynucleotide phosphorylase; ref. 2) on the transferred into these strains (as described above for con- basis of both genetic and biochemical criteria. These include struction of C3/5) to generate strains CAN/20-12 and the enzymes, RNase II (3), RNase D (4), RNase BN (5), and CA265/3-1. A-further derivative of strain CAN 20-12, also RNase R (5, 6). lacking RNase I, was constructed by first isolating a tetracy- Although the in vitro specificities of several of these en- cline-sensitive derivative of the strain (14) and then introduc- zymes have suggested possible functions (2, 3, 5, 7), in no ing rna by cotransduction with zbd-279::TnlO present in case has a physiological role been conclusively demonstrat- strain SK2255 (obtained from the Coli Genetic Stock Center, ed. One in vivo function that is thought to require exoribonu- Yale University). Extracts of this strain, CAN 20-12E, were clease action is the -C-C-A end-turnover of tRNA (8). In an devoid of RNase I, RNase II, RNase D, and RNase BN ac- attempt to determine which, if any, of the known exoribonu- tivity. cleases might be involved in this process, we have examined Derivatives of the various RNase- strains lacking tRNA the state of the 3' terminus in tRNAs isolated from strains nucleotidyltransferase (cca mutants) were constructed by deficient in each of these enzymes (9-11) and also lacking cotransduction of the cca locus from strain 35-10 with toiC tRNA nucleotidyltransferase so that defective -C-C-A se- as described (15). quences would not be repaired (8). To our surprise, in no Strains were grown in YT medium (16), usually supple- case did the absence of an RNase affect the amount of defec- mented with 0.4% glucose. Tetracycline-resistant strains tive tRNA in the double-mutant strains, suggesting that an- were grown in medium containing tetracycline at 10 ,ug per other nucleolytic activity might be involved in the end-turn- ml. over process. Substrates. E. coli tRNA was isolated by phenol extraction Accordingly, we investigated whether an additional exori- and isopropanol fractionation as described (17). tRNA-C-C- bonuclease might be present in extracts of E. coli that could [14C]A, tRNA-C-[14C]U, phosphodiesterase-treated [32p]- remove the terminal AMP residue from tRNA. In this paper tRNA, and [32P]rRNA were isolated or synthesized as re- we describe the identification of this nuclease, termed ported (4, 13, 18). [3H]Poly(A) and nonradioactive poly(A) RNase T, present an initial description of its properties, and were purchased from Miles. Other Materials. Radioactive nucleoside triphosphates The publication costs of this article were defrayed in part by page charge were obtained from Schwarz/Mann. Ultrogel AcA44 was payment. This article must therefore be hereby marked "advertisement" from LKB. Rabbit liver tRNA nucleotidyltransferase was in accordance with 18 U.S.C. §1734 solely to indicate this fact. purified as reported (19). All salts were reagent grade. 4290 Downloaded by guest on September 29, 2021 Biochemistry: Deutscher et aL Proc. Natl. Acad. Sci USA 81 (1984) 4291 Pieparation of Extracts and Gel Filtration. High-speed su- Table 2. Activity against tRNA-C-C-['4C]A in extracts prepared pernatant (S100) fractions for gel filtration were prepared from RNase- strains after rupture of the cells in an Aminco French press as de- tRNA-C-C-[14C]A hydrolysis, scribed (13). In some experiments cells were opened in buff- Strain Relevant phenotype nmol/15 min er B (50 mM glycine-NaOH, pH 9.0/0.1 mM dithiothrei- tol/0.1 mM EDTA). For small-scale experiments, cells were CA265 RNase' 0.47 ruptured by sonication. Gel filtration on Ultrogel AcA44 was CAN RNase BN- 0.44 carried out in 10 mM Tris chloride, pH 7.5/0.1 mM CAN/20 RNase BN-, II 0.41 EDTA/0.1 mM dithiothreitol/0.1 mM phenylmethylsulfonyl CAN/20-12 RNase BN-, II, D- 0.30 fluoride/1 M KCl/10% (vol/vol) glycerol. Cells were grown to A550 of 1, removed from thb growth medium Assays. The level of defective 3' termini in tRNA isolated by centrifugation, and resuspended in one-fourth the volume in 50 from various mutant cells was determined from the amount mM glycine-NaOH (pH 9.0). Cells were ruptured by sonication, and of [14C]AMP that could be incorporated in the presence of cell debris was removed by centrifugation. Aliquots of 100 Al were purified tRNA nucleotidyltransferase as described (20). assayed in 200-1.d reaction mixtures containing 50 mM glycine- Activity of the various RNases was measured under opti- NaOH (pH 9.0), 5 mM MgC12, and 53 ,ug of tRNA-C-C-['4C]A at mal conditions by determination ofacid-soluble radioactivity 370C for 15 min. Acid-soluble radioactivity was measured as de- released from different substrates (4). Standard assays were scribed. carried out in 100-,ul reaction mixtures: 20 mM glycine- zyme that might remove the 3' AMP residue from tRNA. NaOH, pH 8.9/5 mM MgCl2 containing 45 jig of diesterase- Identification of RNase T. To facilitate detection of a dis- treated [32P]tRNA for RNase D; 20 mM Tris chloride, pH tinct RNase that could act upon tRNA-C-C-[14CIA, we pre- 7.5/5 mM MgCl2/100 mM KCl/1 mM [3H]poly(A) for RNase pared extracts from a series of mutant cells, each lacking an II; 20 mM Tris chloride, pH 8.0/0.25 mM MgCl2/300 mM additional RNase compared to its parent (Table 2). The ab- KCl containing '70 ,ug of [32P]rRNA for RNase R; 20 mM sence of RNase BN led to about a 7% decrease in hydrolysis Hepes-NaOH, pH 6.5/0.2 mM CoCl2/200 mM KCI contain- of tRNA-C-C-['4CJA, and a similar decrease was found upon ing 37 ,ug of tRNA-C-['4CJU for RNase BN; and 50 mM gly- the further removal of RNase II. In a strain also lacking cine-NaOH, pH 8.9/5 mM MgCI2 containing 34 ,ug of tRNA- RNase D, an additional 20% of the activity was lost; howev- C-C-[14C]A for RNase T.
Load more

Recommended publications
  • Determination of Pk, Values of the Histidine Side
    Protein Science (1997), 6:1937-1944. Cambridge University Press. Printed in the USA Copyright 0 1997 The Protein Society Determination of pK, values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis TUN LIU, MARGRET RYAN, FREDERICK W. DAHLQUIST, AND 0. HAYES GRIFFITH Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403 (RECEIVEDDecember 4, 1996: ACCEPTEDMay 19, 1997) Abstract Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pK,, values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis. The protein was selec- tively labeled with '3C"-histidine. A series of 'H-I3C HSQC NMR spectra were acquired over a pH range of 4.0-9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 'H-"C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity. The active site histidines, His32 and His82, display pK,, values of 7.6 and 6.9, respectively. His92 and His227 exhibit pK, values of 5.4 and 6.9.
    [Show full text]
  • Families and the Structural Relatedness Among Globular Proteins
    Protein Science (1993), 2, 884-899. Cambridge University Press. Printed in the USA. Copyright 0 1993 The Protein Society -~~ ~~~~ ~ Families and the structural relatedness among globular proteins DAVID P. YEE AND KEN A. DILL Department of Pharmaceutical Chemistry, University of California, San Francisco, California94143-1204 (RECEIVEDJanuary 6, 1993; REVISEDMANUSCRIPT RECEIVED February 18, 1993) Abstract Protein structures come in families. Are families “closely knit” or “loosely knit” entities? We describe a mea- sure of relatedness among polymer conformations. Based on weighted distance maps, this measure differs from existing measures mainly in two respects: (1) it is computationally fast, and (2) it can compare any two proteins, regardless of their relative chain lengths or degree of similarity. It does not require finding relative alignments. The measure is used here to determine the dissimilarities between all 12,403 possible pairs of 158 diverse protein structures from the Brookhaven Protein Data Bank (PDB). Combined with minimal spanning trees and hier- archical clustering methods,this measure is used to define structural families. It is also useful for rapidly searching a dataset of protein structures for specific substructural motifs.By using an analogy to distributions of Euclid- ean distances, we find that protein families are not tightly knit entities. Keywords: protein family; relatedness; structural comparison; substructure searches Pioneering work over the past 20 years has shown that positions after superposition. RMS is a useful distance proteins fall into families of related structures (Levitt & metric for comparingstructures that arenearly identical: Chothia, 1976; Richardson, 1981; Richardson & Richard- for example, when refining or comparing structures ob- son, 1989; Chothia & Finkelstein, 1990).
    [Show full text]
  • Mechanisms of Salmonella Attachment and Survival on In-Shell Black Peppercorns, Almonds, and Hazelnuts
    UC Irvine UC Irvine Previously Published Works Title Mechanisms of Salmonella Attachment and Survival on In-Shell Black Peppercorns, Almonds, and Hazelnuts. Permalink https://escholarship.org/uc/item/5534264q Authors Li, Ye Salazar, Joelle K He, Yingshu et al. Publication Date 2020 DOI 10.3389/fmicb.2020.582202 Peer reviewed eScholarship.org Powered by the California Digital Library University of California fmicb-11-582202 October 19, 2020 Time: 10:46 # 1 ORIGINAL RESEARCH published: 23 October 2020 doi: 10.3389/fmicb.2020.582202 Mechanisms of Salmonella Attachment and Survival on In-Shell Black Peppercorns, Almonds, and Hazelnuts Ye Li1, Joelle K. Salazar2, Yingshu He1, Prerak Desai3, Steffen Porwollik3, Weiping Chu3, Palma-Salgado Sindy Paola4, Mary Lou Tortorello2, Oscar Juarez5, Hao Feng4, Michael McClelland3* and Wei Zhang1* 1 Department of Food Science and Nutrition, Illinois Institute of Technology, Bedford Park, IL, United States, 2 Division of Food Processing Science and Technology, U.S. Food and Drug Administration, Bedford Park, IL, United States, 3 Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA, United States, 4 Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, United States, 5 Department Edited by: of Biology, Illinois Institute of Technology, Chicago, IL, United States Chrysoula C. Tassou, Institute of Technology of Agricultural Products, Hellenic Agricultural Salmonella enterica subspecies I (ssp 1) is the leading cause of hospitalizations and Organization, Greece deaths due to known bacterial foodborne pathogens in the United States and is Reviewed by: frequently implicated in foodborne disease outbreaks associated with spices and nuts.
    [Show full text]
  • Exoribonuclease Nibbler Shapes the 3″ Ends of Micrornas
    Current Biology 21, 1878–1887, November 22, 2011 ª2011 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2011.09.034 Article The 30-to-50 Exoribonuclease Nibbler Shapes the 30 Ends of MicroRNAs Bound to Drosophila Argonaute1 Bo W. Han,1 Jui-Hung Hung,2 Zhiping Weng,2 precursor miRNAs (pre-miRNAs) [8]. Pre-miRNAs comprise Phillip D. Zamore,1,* and Stefan L. Ameres1,* a single-stranded loop and a partially base-paired stem whose 1Howard Hughes Medical Institute and Department of termini bear the hallmarks of RNase III processing: a two-nucle- Biochemistry and Molecular Pharmacology otide 30 overhang, a 50 phosphate, and a 30 hydroxyl group. 2Program in Bioinformatics and Integrative Biology Nuclear pre-miRNAs are exported by Exportin 5 to the cyto- University of Massachusetts Medical School, plasm, where the RNase III enzyme Dicer liberates w22 nt 364 Plantation Street, Worcester, MA 01605, USA mature miRNA/miRNA* duplexes from the pre-miRNA stem [9–12]. Like all Dicer products, miRNA duplexes contain two- nucleotide 30 overhangs, 50 phosphate, and 30 hydroxyl groups. Summary In flies, Dicer-1 cleaves pre-miRNAs to miRNAs, whereas Dicer-2 converts long double-stranded RNA (dsRNA) into Background: MicroRNAs (miRNAs) are w22 nucleotide (nt) small interfering RNAs (siRNAs), which direct RNA interference small RNAs that control development, physiology, and pathol- (RNAi), a distinct small RNA silencing pathway required for ogy in animals and plants. Production of miRNAs involves the host defense against viral infection and somatic transposon sequential processing of primary hairpin-containing RNA poly- mobilization, as well as gene silencing triggered by exogenous merase II transcripts by the RNase III enzymes Drosha in the dsRNA [13, 14].
    [Show full text]
  • Supplementary Materials
    Supplementary Materials COMPARATIVE ANALYSIS OF THE TRANSCRIPTOME, PROTEOME AND miRNA PROFILE OF KUPFFER CELLS AND MONOCYTES Andrey Elchaninov1,3*, Anastasiya Lokhonina1,3, Maria Nikitina2, Polina Vishnyakova1,3, Andrey Makarov1, Irina Arutyunyan1, Anastasiya Poltavets1, Evgeniya Kananykhina2, Sergey Kovalchuk4, Evgeny Karpulevich5,6, Galina Bolshakova2, Gennady Sukhikh1, Timur Fatkhudinov2,3 1 Laboratory of Regenerative Medicine, National Medical Research Center for Obstetrics, Gynecology and Perinatology Named after Academician V.I. Kulakov of Ministry of Healthcare of Russian Federation, Moscow, Russia 2 Laboratory of Growth and Development, Scientific Research Institute of Human Morphology, Moscow, Russia 3 Histology Department, Medical Institute, Peoples' Friendship University of Russia, Moscow, Russia 4 Laboratory of Bioinformatic methods for Combinatorial Chemistry and Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia 5 Information Systems Department, Ivannikov Institute for System Programming of the Russian Academy of Sciences, Moscow, Russia 6 Genome Engineering Laboratory, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia Figure S1. Flow cytometry analysis of unsorted blood sample. Representative forward, side scattering and histogram are shown. The proportions of negative cells were determined in relation to the isotype controls. The percentages of positive cells are indicated. The blue curve corresponds to the isotype control. Figure S2. Flow cytometry analysis of unsorted liver stromal cells. Representative forward, side scattering and histogram are shown. The proportions of negative cells were determined in relation to the isotype controls. The percentages of positive cells are indicated. The blue curve corresponds to the isotype control. Figure S3. MiRNAs expression analysis in monocytes and Kupffer cells. Full-length of heatmaps are presented.
    [Show full text]
  • Characterization of the Mammalian RNA Exonuclease 5/NEF-Sp As a Testis-Specific Nuclear 3′′′′′ → 5′′′′′ Exoribonuclease
    Downloaded from rnajournal.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Characterization of the mammalian RNA exonuclease 5/NEF-sp as a testis-specific nuclear 3′′′′′ → 5′′′′′ exoribonuclease SARA SILVA,1,2 DAVID HOMOLKA,1 and RAMESH S. PILLAI1 1Department of Molecular Biology, University of Geneva, CH-1211 Geneva 4, Switzerland 2European Molecular Biology Laboratory, Grenoble Outstation, 38042, France ABSTRACT Ribonucleases catalyze maturation of functional RNAs or mediate degradation of cellular transcripts, activities that are critical for gene expression control. Here we identify a previously uncharacterized mammalian nuclease family member NEF-sp (RNA exonuclease 5 [REXO5] or LOC81691) as a testis-specific factor. Recombinant human NEF-sp demonstrates a divalent metal ion-dependent 3′′′′′ → 5′′′′′ exoribonuclease activity. This activity is specific to single-stranded RNA substrates and is independent of their length. The presence of a 2′′′′′-O-methyl modification at the 3′′′′′ end of the RNA substrate is inhibitory. Ectopically expressed NEF-sp localizes to the nucleolar/nuclear compartment in mammalian cell cultures and this is dependent on an amino-terminal nuclear localization signal. Finally, mice lacking NEF-sp are viable and display normal fertility, likely indicating overlapping functions with other nucleases. Taken together, our study provides the first biochemical and genetic exploration of the role of the NEF-sp exoribonuclease in the mammalian genome. Keywords: NEF-sp; LOC81691; Q96IC2; REXON; RNA exonuclease 5; REXO5; 2610020H08Rik INTRODUCTION clease-mediated processing to create their final 3′ ends: poly(A) tails of most mRNAs or the hairpin structure of Spermatogenesis is the process by which sperm cells are replication-dependent histone mRNAs (Colgan and Manley produced in the male germline.
    [Show full text]
  • Genomics of an Extreme Psychrophile, Psychromonas
    BMC Genomics BioMed Central Research article Open Access Genomics of an extreme psychrophile, Psychromonas ingrahamii Monica Riley*1, James T Staley2, Antoine Danchin3, Ting Zhang Wang3, Thomas S Brettin4, Loren J Hauser5, Miriam L Land5 and Linda S Thompson4 Address: 1Bay Paul Center, Marine Biological Laboratory, Woods Hole, MA 02543, USA, 2University of Washington, Seattle, WA 98195-7242, USA, 3Genetics of Bacterial Genomes, CNRS URA2171, Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, France, 4DOE Joint Genome Institute, Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA and 5Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA Email: Monica Riley* - [email protected]; James T Staley - [email protected]; Antoine Danchin - [email protected]; Ting Zhang Wang - [email protected]; Thomas S Brettin - [email protected]; Loren J Hauser - [email protected]; Miriam L Land - [email protected]; Linda S Thompson - [email protected] * Corresponding author Published: 6 May 2008 Received: 3 September 2007 Accepted: 6 May 2008 BMC Genomics 2008, 9:210 doi:10.1186/1471-2164-9-210 This article is available from: http://www.biomedcentral.com/1471-2164/9/210 © 2008 Riley et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The genome sequence of the sea-ice bacterium Psychromonas ingrahamii 37, which grows exponentially at -12C, may reveal features that help to explain how this extreme psychrophile is able to grow at such low temperatures.
    [Show full text]
  • United States Patent (19) 11) 4,039,382 Thang Et Al
    United States Patent (19) 11) 4,039,382 Thang et al. 45 Aug. 2, 1977 54 MMOBILIZED RIBONUCLEASE AND -Enzyme Systems, Journal of Food Science, vol. 39, ALKALINE PHOSPHATASE 1974, (pp. 647-652). 75 Inventors: Minh-Nguy Thang, Bagneux; Annie Zaborsky, O., Immobilized Enzymes, CRC Press, Guissani born Trachtenberg, Fresnes, Cleveland, Ohio, 1973, (pp. 124-126). both of France Primary Examiner-David M. Naff 73 Assignee: Choay S. A., Paris, France Attorney, Agent, or Firm-Browdy and Neimark 21 Appl. No.: 678,459 22 Filed: Apr. 19, 1976 57 ABSTRACT An insoluble, solid matrix carrying simultaneously sev 30 Foreign Application Priority Data eral different enzymatic functions, is constituted by the Apr. 23, 1975 France ................................ 75.12667 conjoint association by irreversible binding on a previ ously activated matrix support, of a nuclease selected 51) int. Cl? ........................... C07G 7/02; C12B 1/00 from the group of ribonucleases A, T, T, U, and an 52 U.S. Cl. ................................... 195/28 N; 195/63; alkaline phosphatease. Free activated groups of the 195/68; 195/DIG. 11; 195/116 matrix after binding of the enzymes, are neutralized by 58 Field of Search ................... 195/63, 68, DIG. 11, a free amino organic base. The support is selected from 195/116, 28 N among non-denaturing supports effecting the irrevers 56) References Cited ible physical adsorption of the enzymes, such as sup ports of glass or quartz beads, highly cross-linked gels PUBLICATIONS of the agarose or cellulose type. Polymers AUott, A. Lee, J. C., Preparation and Properties of Water Insolu Cott, AGot and/or oligonucleotides U, C, A or G, of ble Derivatives of Ribonuclease Ti.
    [Show full text]
  • Apr 1 0 1981 Ubraries 2
    Mechanism of DNA Chain Initiation by the dnaG Protein of Escherichia coli by Daniel Jeffrey Capon B.S., Massachusetts Institute of Technology (1976) SUBMITTED IN PARTIAL FULFILL1ENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY January 1981 0) Massachusetts Institute of Technology 1981 Signature of Author__ %A~A(Departjent of Biology Certified by A U Thesis Supervisor Accepted by ~'RCHIVES Chairman, Departmental Committee MASSACHUSM INSTiTUTE OF TECHNOL1Y APR 1 0 1981 UBRARIES 2 Mechanism of DNA Chain Initiation by the dnaG protein of Escherichia coli by Daniel Jeffrey Capon Submitted to the Department of Biology on January 28, 1981 in partial fulfillment of the requirements for the degree of Doctor of Philosophy ABSTRACT All known DNA polymerases are unable to initiate the synthesis of DNA chains de novo, but are capable of extending the 3' hydroxy terminus of a preexisting 'primer' chain stably annealed to the template strand. The report that partially purified preparations of the dnaG protein, a gene product essential to the replication of E. coli, synthesize RNA primers on phage G4 single-stranded DNA (Bouche, Zechel and Kornberg, 1975) stimulated an investigation into the properties of this enzyme. A thermolabile dnaG protein, prepared from a temperature-sensitive strain of E. coli, was utilized to demonstrate that the ability to prime DNA synthelsis on phage G4 and 0x174 single-stranded DNA resides with the dnaG protein, and that the priming event may be separated from subsequent DNA synthesis. Priming on G4 DNA absolutely requires the E. coli DNA binding protein.
    [Show full text]
  • BRCA1 Binds TERRA RNA and Suppresses R-Loop-Based Telomeric DNA Damage ✉ Jekaterina Vohhodina 1,2 , Liana J
    ARTICLE https://doi.org/10.1038/s41467-021-23716-6 OPEN BRCA1 binds TERRA RNA and suppresses R-Loop-based telomeric DNA damage ✉ Jekaterina Vohhodina 1,2 , Liana J. Goehring1, Ben Liu1,2, Qing Kong1,2, Vladimir V. Botchkarev Jr.1,2, Mai Huynh1, Zhiqi Liu1, Fieda O. Abderazzaq1,2, Allison P. Clark1,2, Scott B. Ficarro1,3,4,5, Jarrod A. Marto 1,3,4,5, ✉ Elodie Hatchi 1,2 & David M. Livingston 1,2 R-loop structures act as modulators of physiological processes such as transcription termi- 1234567890():,; nation, gene regulation, and DNA repair. However, they can cause transcription-replication conflicts and give rise to genomic instability, particularly at telomeres, which are prone to forming DNA secondary structures. Here, we demonstrate that BRCA1 binds TERRA RNA, directly and physically via its N-terminal nuclear localization sequence, as well as telomere- specific shelterin proteins in an R-loop-, and a cell cycle-dependent manner. R-loop-driven BRCA1 binding to CpG-rich TERRA promoters represses TERRA transcription, prevents TERRA R-loop-associated damage, and promotes its repair, likely in association with SETX and XRN2. BRCA1 depletion upregulates TERRA expression, leading to overly abundant TERRA R-loops, telomeric replication stress, and signs of telomeric aberrancy. Moreover, BRCA1 mutations within the TERRA-binding region lead to an excess of TERRA-associated R- loops and telomeric abnormalities. Thus, normal BRCA1/TERRA binding suppresses telomere-centered genome instability. 1 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA. 2 Department of Genetics, Harvard Medical School, Boston, MA, USA. 3 Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USA.
    [Show full text]
  • Coulombic Interactions in Ribonuclease A
    COULOMBIC INTERACTIONS IN RIBONUCLEASE A by Barbra Mindy Templer A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biochemistry) at the UNIVERSITY OF WISCONSIN-MADISON 1998 A dissertation entitled Coulombic Interactions in Ribonuclease A submitted to the Graduate School of the University of Wisconsin-Madison in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Barbra Mindy Templer Date of Final Oral Examination: May 21, 1998 1998 Month & Year Degree to be awarded: December May August * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * • * * * * * * * * * * * * * * * * * * * * * * Approval Signal ures of Dissertation Readers: Signature, Dean of Graduate School l---[-;:-~ ,,(1~ h~~~ ~~~ ~ /tYk ,ec,~~ COULOMBIC INTERACTIONS IN RIBONUCLEASE A Barbra Mindy Templer Under the supervision of Associate Professor Ronald T. Raines At the University of Wisconsin-Madison The interactions between bovine pancreatic ribonuclease (RNase) A and its RNA substrate extend beyond the scissile P-Os' bond. Enzymic subsites interact with the bases and phosphoryl groups of the substrate. Those residues interacting with the phosphoryl groups comprise the PO, PI, and P2 subsites, with the scissile bond residing in the PI subsite. In this Dissertation, the function of the PO and P2 subsites of RNase A is characterized in detail. A new subsite, P( -1), is also described. Lys66 (PO subsite) and Lys7 and Argl0 (P2 subsite) were replaced with alanine residues. Wild-type RNase A and the K66A, K7 AJRlOA, and K7 AJRlONK66A variants were evaluated as catalysts for the cleavage of poly(cytidylic acid) and for their abilities to bind to single-stranded DNA, a substrate analog (Chapter Two). The values of kcal' Km, and kcalKm for poly(C) cleavage were affected by altering the PO and P2 subsites as were the values of Kd for RNase A-d(AUAA.) complex formation.
    [Show full text]
  • Role for Cytoplasmic Nucleotide Hydrolysis in Hepatic Function and Protein Synthesis
    Role for cytoplasmic nucleotide hydrolysis in hepatic function and protein synthesis Benjamin H. Hudson1, Joshua P. Frederick1, Li Yin Drake, Louis C. Megosh, Ryan P. Irving, and John D. York2,3 Department of Pharmacology and Cancer Biology, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710 Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved February 11, 2013 (received for review March 26, 2012) Nucleotide hydrolysis is essential for many aspects of cellular (5–7), which is degraded to 5′-AMP by a family of enzymes known function. In the case of 3′,5′-bisphosphorylated nucleotides, mam- as 3′-nucleotidases (8). mals possess two related 3′-nucleotidases, Golgi-resident 3′-phos- Mammalian genomes encode two 3′-nucleotidases, the recently phoadenosine 5′-phosphate (PAP) phosphatase (gPAPP) and characterized Golgi-resident PAP phosphatase (gPAPP) and Bisphosphate 3′-nucleotidase 1 (Bpnt1). gPAPP and Bpnt1 localize Bisphosphate 3′-nucleotidase 1 (Bpnt1), which localize to the to distinct subcellular compartments and are members of a con- Golgi lumen and cytoplasm, respectively (Fig. 1B)(8–11). gPAPP served family of metal-dependent lithium-sensitive enzymes. Al- and Bpnt1 are members of a family of small-molecule phospha- though recent studies have demonstrated the importance of tases whose activities are both dependent on divalent cations and gPAPP for proper skeletal development in mice and humans, the inhibited by lithium (8). The family comprises seven mammalian role of Bpnt1 in mammals remains largely unknown. Here we re- gene products: fructose bisphosphatase 1 and 2, inositol monophos- port that mice deficient for Bpnt1 do not exhibit skeletal defects phatase 1 and 2, inositol polyphosphate 1-phosphatase, gPAPP, but instead develop severe liver pathologies, including hypopro- and Bpnt1 (Fig.
    [Show full text]