Method for Rapid Analysis of Mutant RNA Polymerase Activity on Templates Containing Unnatural Nucleotides
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A Simple Colorimetric RNA Polymerase Assay
Virology 274, 429–437 (2000) doi:10.1006/viro.2000.0492, available online at http://www.idealibrary.com on Exploiting Polymerase Promiscuity: A Simple Colorimetric RNA Polymerase Assay William Vassiliou,* Jeffery B. Epp,† Bin-Bin Wang,* Alfred M. Del Vecchio,‡,1 Theodore Widlanski,§ and C. Cheng Kao*,2 *Department of Biology, Indiana University, Bloomington, Indiana 47405; †Dow AgroSciences, Indianapolis, Indiana 46268; ‡SmithKline Beecham Inc., Collegeville, Pennsylvania 19426; and §Department of Chemistry, Indiana University, Bloomington, Indiana 47405 Received May 18, 2000; accepted June 29, 2000 We developed a convenient colorimetric assay for monitoring RNA synthesis from DNA-dependent RNA polymerases (DdRp) and viral RNA-dependent RNA polymerases (RdRp). ATP and GTP with a p-nitrophenyl moiety attached to the ␥-phosphate were synthesized (PNP–NTPs). These PNP–NTPs can be used for RNA synthesis by several RNA polymerases, including the RdRps from brome mosaic virus and bovine viral diarrhea virus and the DdRps from bacteriophage T7 and SP6. When the polymerase reactions were performed in the presence of alkaline phosphatase, which digests the p-nitrophe- nylpyrophosphate side-product of phosphoryl transfer to the chromogenic p-nitrophenylate, an increase in absorbence at 405 nm was observed. These nucleotide analogues were used in continuous colorimetric monitoring of polymerase activity. Furthermore, the PNP–NTPs were found to be stable and utilized by RNA polymerases in the presence of human plasma. This simple colorimetric polymerase assay can be performed in a standard laboratory spectrophotometer and will be useful in screens for inhibitors of viral RNA synthesis. © 2000 Academic Press INTRODUCTION polymerases are primarily tested using radioactive assays that require special handling of the isotope RNA and DNA polymerases carry out the synthesis of and are incompatible with continuous monitoring of oligonucleotides by transfer of a nucleoside monophos- polymerase activity (Ferrari et al., 1999). -
(12) United States Patent (10) Patent No.: US 9,598.458 B2 Shimizu Et Al
USO09598458B2 (12) United States Patent (10) Patent No.: US 9,598.458 B2 Shimizu et al. (45) Date of Patent: Mar. 21, 2017 (54) ASYMMETRICAUXILLARY GROUP 3,687,808 A 8/1972 Merigan et al. 3,745,162 A 7/1973 Helsley 4,022,791 A 5/1977 Welch, Jr. (71) Applicant: NYTE SCIENCES JAPAN, 4,113,869 A 9, 1978 Gardner ... Kagoshima-shi (JP) 4.415,732 A 1 1/1983 Caruthers et al. 4,458,066 A 7/1984 Caruthers et al. (72) Inventors: Mamoru Shimizu, Uruma (JP); 4,500,707 A 2f1985 Caruthers et al. Takeshi Wada, Kashiwa (JP) 4,542,142 A 9, 1985 Martel et al. 4,659,774 A 4, 1987 Webb et al. 4,663,328 A 5, 1987 Lafon (73) Assignee: YESCIENCES JAPAN, 4,668,777. A 5/1987 Caruthers et al. ... Kagoshima-shi (JP) 4,725,677 A 2/1988 Koster et al. 4,735,949 A 4, 1988 Domagala et al. (*) Notice: Subject to any disclaimer, the term of this 4,840,956 A 6/1989 Domagala et al. patent is extended or adjusted under 35 3:38 A . 3. EthOester Phet al. et al. U.S.C. 154(b) by 17 days. 4,973,679 A 1 1/1990 Caruthers et al. 4,981,957 A 1/1991 Lebleu et al. (21) Appl. No.: 14/414,604 5,047,524. A 9/1991 Andrus et al. 5,118,800 A 6/1992 Smith et al. (22) PCT Filed: Jul. 12, 2013 5,130,302 A 7/1992 Spielvogel et al. -
A Mutation in DNA Polymerase Α Rescues WEE1KO Sensitivity to HU
International Journal of Molecular Sciences Article A Mutation in DNA Polymerase α Rescues WEE1KO Sensitivity to HU Thomas Eekhout 1,2 , José Antonio Pedroza-Garcia 1,2 , Pooneh Kalhorzadeh 1,2, Geert De Jaeger 1,2 and Lieven De Veylder 1,2,* 1 Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; [email protected] (T.E.); [email protected] (J.A.P.-G.); [email protected] (P.K.); [email protected] (G.D.J.) 2 Center for Plant Systems Biology, VIB, 9052 Gent, Belgium * Correspondence: [email protected] Abstract: During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication and are hypersensitive to replication stress. Here, we report the identification of the pola-2 mutant, mutated in the catalytic subunit of DNA polymerase α, as a suppressor mutant of wee1. The mutated protein appears to be less stable, causing a loss of interaction with its subunits and resulting in a prolonged S-phase. Keywords: replication stress; DNA damage; cell cycle checkpoint Citation: Eekhout, T.; Pedroza- 1. Introduction Garcia, J.A.; Kalhorzadeh, P.; De Jaeger, G.; De Veylder, L. A Mutation DNA replication is a highly complex process that ensures the chromosomes are in DNA Polymerase α Rescues correctly replicated to be passed onto the daughter cells during mitosis. Replication starts WEE1KO Sensitivity to HU. Int. -
Excited State Dynamics of Isocytosine; a Hybrid Case of Canonical Nucleobase Photodynamics
The Journal of Physical Chemistry Letters This document is confidential and is proprietary to the American Chemical Society and its authors. Do not copy or disclose without written permission. If you have received this item in error, notify the sender and delete all copies. Excited State Dynamics of Isocytosine; a Hybrid Case of Canonical Nucleobase Photodynamics Journal: The Journal of Physical Chemistry Letters Manuscript ID jz-2017-020322.R1 Manuscript Type: Letter Date Submitted by the Author: n/a Complete List of Authors: Berenbeim, Jacob; UC Santa Barbara, Chemistry and Biochemistry Boldissar, Samuel; UCSB, Department of Chemistry Siouri, Faady; UCSB, Department of Chemistry Gate, Gregory; UCSB, Department of Chemistry Haggmark, Michael; UC Santa Barbara, Chemistry and Biochemistry Aboulache, Briana; University of California Santa Barbara Cohen, Trevor; UC Santa Barbara, Chemistry and Biochemistry De Vries, Mattanjah; UCSB, Department of Chemistry ACS Paragon Plus Environment Page 1 of 12 The Journal of Physical Chemistry Letters 1 2 3 4 Excited State Dynamics of Isocytosine; A Hybrid Case of Canonical 5 Nucleobase Photodynamics 6 7 Jacob A. Berenbeim, Samuel Boldissar, Faady M. Siouri, Gregory Gate, Michael R. 8 Haggmark, Briana Aboulache, Trevor Cohen, and Mattanjah S. de Vries* 9 10 Department of Chemistry and Biochemistry, University of California Santa 11 12 Barabara, CA 93106-9510 13 *E-mail: [email protected] 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 ACS Paragon Plus Environment The Journal of Physical Chemistry Letters Page 2 of 12 1 2 3 Abstract 4 5 We present resonant two-photon ionization (R2PI) spectra of isocytosine (isoC) and pump-probe 6 results on two of its tautomers. -
The Conserved Structure of Plant Telomerase RNA Provides the Missing Link for an Evolutionary Pathway from Ciliates to Humans
The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans Jiarui Songa, Dhenugen Logeswaranb,1, Claudia Castillo-Gonzáleza,1, Yang Lib, Sreyashree Bosea, Behailu Birhanu Aklilua, Zeyang Mac,d, Alexander Polkhovskiya,e, Julian J.-L. Chenb,2, and Dorothy E. Shippena,2 aDepartment of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843; bSchool of Molecular Sciences, Arizona State University, Tempe, AZ 85287; cNational Maize Improvement Center of China, China Agricultural University, 100193 Beijing, China; dCollege of Agronomy and Biotechnology, China Agricultural University, 100193 Beijing, China; and eCenter of Life Sciences, Skolkovo Institute of Science and Technology, 121205 Moscow, Russian Federation Edited by Thomas R. Cech, University of Colorado Boulder, Boulder, CO, and approved October 24, 2019 (received for review September 4, 2019) Telomerase is essential for maintaining telomere integrity. Although transcribed by RNA polymerase III (Pol III) (6, 7). The La-related telomerase function is widely conserved, the integral telomerase protein P65 in Tetrahymena recognizes the 3′ poly-U tail of TR RNA (TR) that provides a template for telomeric DNA synthesis has and bends the RNA to facilitate telomerase RNP assembly (8, 9). diverged dramatically. Nevertheless, TR molecules retain 2 highly In contrast, fungi maintain much larger TR molecules (900 to conserved structural domains critical for catalysis: a template- 2,400 nt) that are transcribed by RNA polymerase II (Pol II) (3). proximal pseudoknot (PK) structure and a downstream stem-loop The 3′ end maturation of fungal TRs requires components of the structure. Here we introduce the authentic TR from the plant canonical snRNA biogenesis pathway and results in RNP assembly Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. -
Yeast Genome Gazetteer P35-65
gazetteer Metabolism 35 tRNA modification mitochondrial transport amino-acid metabolism other tRNA-transcription activities vesicular transport (Golgi network, etc.) nitrogen and sulphur metabolism mRNA synthesis peroxisomal transport nucleotide metabolism mRNA processing (splicing) vacuolar transport phosphate metabolism mRNA processing (5’-end, 3’-end processing extracellular transport carbohydrate metabolism and mRNA degradation) cellular import lipid, fatty-acid and sterol metabolism other mRNA-transcription activities other intracellular-transport activities biosynthesis of vitamins, cofactors and RNA transport prosthetic groups other transcription activities Cellular organization and biogenesis 54 ionic homeostasis organization and biogenesis of cell wall and Protein synthesis 48 plasma membrane Energy 40 ribosomal proteins organization and biogenesis of glycolysis translation (initiation,elongation and cytoskeleton gluconeogenesis termination) organization and biogenesis of endoplasmic pentose-phosphate pathway translational control reticulum and Golgi tricarboxylic-acid pathway tRNA synthetases organization and biogenesis of chromosome respiration other protein-synthesis activities structure fermentation mitochondrial organization and biogenesis metabolism of energy reserves (glycogen Protein destination 49 peroxisomal organization and biogenesis and trehalose) protein folding and stabilization endosomal organization and biogenesis other energy-generation activities protein targeting, sorting and translocation vacuolar and lysosomal -
Human Glucokinase Gene
Proc. Nati. Acad. Sci. USA Vol. 89, pp. 7698-7702, August 1992 Genetics Human glucokinase gene: Isolation, characterization, and identification of two missense mutations linked to early-onset non-insulin-dependent (type 2) diabetes mellitus (glucose/metabolism/phosphorylation/structure4unctlon/chromosome 7) M. STOFFEL*, PH. FROGUELt, J. TAKEDA*, H. ZOUALItt, N. VIONNET*, S. NISHI*§, I. T. WEBER¶, R. W. HARRISON¶, S. J. PILKISII, S. LESAGEtt, M. VAXILLAIREtt, G. VELHOtt, F. SUNtt, F. lIRSt, PH. PASSAt, D. COHENt, AND G. I. BELL*"** *Howard Hughes Medical Institute, and Departments of Biochemistry and Molecular Biology, and of Medicine, The University of Chicago, 5841 South Maryland Avenue, MC1028, Chicago, IL 60637; §Second Division of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-32, Japan; IDepartment of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107; IlDepartment of Physiology and Biophysics, State University of New York, Stony Brook, NY 11794; tCentre d'Etude du Polymorphisme Humain, 27 rue Juliette Dodu, and Service d'Endocrinologie, H6pital Saint-Louis, 75010 Paris, France; and tG6ndthon, 1 rue de l'Internationale, 91000 Evry, France Communicated by Jean Dausset, May 28, 1992 ABSTRACT DNA polymorphisms in the glucokinase gene by maintaining a gradient for glucose transport into these cells have recently been shown to be tightly linked to early-onset thereby regulating hepatic glucose disposal. In (3 cells, glu- non-insulin-dependent diabetes mellitus in "80% of French cokinase is believed to be part of the glucose-sensing mech- families with this form of diabetes. We previously identified a anism and to be involved in the regulation ofinsulin secretion. -
5-Fluorocytosine/Isocytosine Monohydrate. the First Example of Isomorphic and Isostructural Co-Crystal of Pyrimidine Nucleobases
crystals Article 5-Fluorocytosine/Isocytosine Monohydrate. The First Example of Isomorphic and Isostructural Co-Crystal of Pyrimidine Nucleobases Gustavo Portalone Department of Chemistry, ‘Sapienza’ University of Rome, 00185 Rome, Italy; [email protected]; Tel.: +396-4991-3715 Received: 1 October 2020; Accepted: 2 November 2020; Published: 3 November 2020 Abstract: To date, despite the crucial role played by cytosine, uracil, and thymine in the DNA/RNA replication process, no examples showing isomorphic and isostructural behavior among binary co-crystals of natural or modified pyrimidine nucleobases have been so far reported in the literature. In view of the relevance of biochemical and pharmaceutical compounds such as pyrimidine nucleobases and their 5-fluoroderivatives, co-crystals of the molecular complex formed by 5-fluorocytosine and isocytosine monohydrate, C H FN O C H N O H O, have been synthesized 4 4 3 · 4 5 3 · 2 by a reaction between 5-fluorocytosine and isocytosine. They represent the first example of isomorphic and isostructural binary co-crystals of pyrimidine nucleobases, as X-ray diffraction analysis shows structural similarities in the solid-state organization of molecules with that of the (1:1) 5-fluorocytosine/5-fluoroisocytosine monohydrate molecular complex, which differs solely in the H/F substitution at the C5 position of isocytosine. Molecules of 5-fluorocytosine and isocytosine are present in the crystal as 1H and 3H-ketoamino tautomers, respectively. They form almost coplanar WC base pairs through nucleobase-to-nucleobase DAA/ADD hydrogen bonding interactions, demonstrating that complementary binding enables the crystallization of specific tautomers. Additional peripheral hydrogen bonds involving all available H atom donor and acceptor sites of the water molecule give a three-dimensional polymeric structure. -
Arthur Kornberg Discovered (The First) DNA Polymerase Four
Arthur Kornberg discovered (the first) DNA polymerase Using an “in vitro” system for DNA polymerase activity: 1. Grow E. coli 2. Break open cells 3. Prepare soluble extract 4. Fractionate extract to resolve different proteins from each other; repeat; repeat 5. Search for DNA polymerase activity using an biochemical assay: incorporate radioactive building blocks into DNA chains Four requirements of DNA-templated (DNA-dependent) DNA polymerases • single-stranded template • deoxyribonucleotides with 5’ triphosphate (dNTPs) • magnesium ions • annealed primer with 3’ OH Synthesis ONLY occurs in the 5’-3’ direction Fig 4-1 E. coli DNA polymerase I 5’-3’ polymerase activity Primer has a 3’-OH Incoming dNTP has a 5’ triphosphate Pyrophosphate (PP) is lost when dNMP adds to the chain E. coli DNA polymerase I: 3 separable enzyme activities in 3 protein domains 5’-3’ polymerase + 3’-5’ exonuclease = Klenow fragment N C 5’-3’ exonuclease Fig 4-3 E. coli DNA polymerase I 3’-5’ exonuclease Opposite polarity compared to polymerase: polymerase activity must stop to allow 3’-5’ exonuclease activity No dNTP can be re-made in reversed 3’-5’ direction: dNMP released by hydrolysis of phosphodiester backboneFig 4-4 Proof-reading (editing) of misincorporated 3’ dNMP by the 3’-5’ exonuclease Fidelity is accuracy of template-cognate dNTP selection. It depends on the polymerase active site structure and the balance of competing polymerase and exonuclease activities. A mismatch disfavors extension and favors the exonuclease.Fig 4-5 Superimposed structure of the Klenow fragment of DNA pol I with two different DNAs “Fingers” “Thumb” “Palm” red/orange helix: 3’ in red is elongating blue/cyan helix: 3’ in blue is getting edited Fig 4-6 E. -
Taming the Wild Rubisco: Explorations in Functional Metagenomics
Taming the Wild RubisCO: Explorations in Functional Metagenomics DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Brian Hurin Witte, M.S. Graduate Program in Microbiology The Ohio State University 2012 Dissertation Committee : F. Robert Tabita, Advisor Joseph Krzycki Birgit E. Alber Paul Fuerst Copyright by Brian Hurin Witte 2012 Abstract Ribulose bisphosphate carboxylase/oxygenase (E.C. 4.1.1.39) (RubisCO) is the most abundant protein on Earth and the mechanism by which the vast majority of carbon enters the planet’s biosphere. Despite decades of study, many significant questions about this enzyme remain unanswered. As anthropogenic CO2 levels continue to rise, understanding this key component of the carbon cycle is crucial to forecasting feedback circuits, as well as to engineering food and fuel crops to produce more biomass with few inputs of increasingly scarce resources. This study demonstrates three means of investigating the natural diversity of RubisCO. Chapter 1 builds on existing DNA sequence-based techniques of gene discovery and shows that RubisCO from uncultured organisms can be used to complement growth in a RubisCO-deletion strain of autotrophic bacteria. In a few short steps, the time-consuming work of bringing an autotrophic organism in to pure culture can be circumvented. Chapter 2 details a means of entirely bypassing the bias inherent in sequence-based gene discovery by using selection of RubisCO genes from a metagenomic library. Chapter 3 provides a more in-depth study of the RubisCO from the methanogenic archaeon Methanococcoides burtonii. -
RNA-Dependent RNA Polymerase Speed and Fidelity Are Not the Only Determinants of the Mechanism Or Efficiency of Recombination
G C A T T A C G G C A T genes Article RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination Hyejeong Kim y, Victor D. Ellis III, Andrew Woodman, Yan Zhao, Jamie J. Arnold y and , Craig E. Cameron y * Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 201 Althouse Laboratory, University Park, PA 16802, USA; [email protected] (H.K.); [email protected] (V.D.E.); [email protected] (A.W.); [email protected] (Y.Z.); [email protected] (J.J.A.) * Correspondence: [email protected]; Tel.: +1-919-966-9699 Present address: Department of Microbiology and Immunology, School of Medicine, University of North y Carolina at Chapel Hill, 125 Mason Farm Rd., Chapel Hill, NC 27599-7290, USA. Received: 15 October 2019; Accepted: 21 November 2019; Published: 25 November 2019 Abstract: Using the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the context of the KH virus, RdRp-coding sequence evolves, selecting for the following substitutions: I331F (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH being competent for forced-copy-choice recombination. -
Durham Research Online
Durham Research Online Deposited in DRO: 04 January 2019 Version of attached le: Published Version Peer-review status of attached le: Peer-reviewed Citation for published item: Pohl, Radek and Socha, Ond§rejand Slav¡§cek,Petr and S¡ala,Michal§ and Hodgkinson, Paul and Dra§c¡nsk¡y, Martin (2018) 'Proton transfer in guaninecytosine base pair analogues studied by NMR spectroscopy and PIMD simulations.', Faraday discussions., 212 . pp. 331-344. Further information on publisher's website: https://doi.org/10.1039/C8FD00070K Publisher's copyright statement: This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Additional information: Use policy The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that: • a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders. Please consult the full DRO policy for further details. Durham University Library, Stockton Road, Durham DH1 3LY, United Kingdom Tel : +44 (0)191 334 3042 | Fax : +44 (0)191 334 2971 https://dro.dur.ac.uk Faraday Discussions Cite this: Faraday Discuss.,2018,212,331 View Article Online PAPER View Journal | View Issue Proton transfer in guanine–cytosine base pair analogues studied by NMR spectroscopy and PIMD simulations† a a b a Radek Pohl, Ondˇrej Socha, Petr Slav´ıcek,ˇ Michal Sˇala,´ c a Paul Hodgkinson and Martin Dracˇ´ınsky´ * Received 28th March 2018, Accepted 1st May 2018 DOI: 10.1039/c8fd00070k It has been hypothesised that proton tunnelling between paired nucleobases significantly enhances the formation of rare tautomeric forms and hence leads to errors in DNA Creative Commons Attribution-NonCommercial 3.0 Unported Licence.