DOCSLIB.ORG

Open Mohammad Almishwat MS 2013

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Open Mohammad Almishwat MS 2013 The Pennsylvania State University The Graduate School Eberly College of Science TIME-RESOLVED X-RAY CRYSTALLOGRAPHY OF DNA POLYMERASES: A PLATFORM FOR STUDIES IN STRUCTURAL AND MECHANISTIC ENZYMOLOGY A Thesis in Biochemistry, Microbiology, and Molecular Biology by Mohammad A. Almishwat © 2013 Mohammad A. Almishwat Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2013 The thesis of Mohammad A. Almishwat was reviewed and approved* by the following: Katsuhiko Murakami Associate Professor of Biochemistry and Molecular Biology Thesis Co-Adviser J. Gregory Ferry Stanley Person Professor of Biochemistry and Molecular Biology Thesis Co-Adviser Susan Hafenstein Assistant Professor of Medicine, Microbiology and Immunology Paul Babitzke Professor of Biochemistry and Molecular Biology Co-Director of Graduate Studies *Signatures are on file in the Graduate School. ii Abstract DNA-dependent DNA polymerase is the enzyme responsible for carrying out DNA replication. DNA polymerase regulates the faithful transmission of an organism’s genetic material, and performs various tasks involved in correcting errors that occur during the process of DNA replication. Extensive genetic and biochemical research has been done to characterize the general mechanism of DNA replication and the factors that govern its fidelity, and yet the mechanistic details of this fidelity, how a correct deoxynucleotide is selected for by the enzyme for incorporation, and an incorrect one is excluded, is yet to be determined. Structural studies of various DNA polymerases and their complexes with DNA have provided a great deal of insight into how catalysis in nucleotide incorporation occurs, and has also provided empirical models of how fidelity is brought about in these enzymes during DNA replication. This thesis describes ongoing work to elucidate the mechanism of high-fidelity nucleotide incorporation during DNA replication by an A-family DNA polymerase, employing biochemical and structural studies to dissect, step-by-step, the structural changes that occur during nucleotide binding and phosphodiester bond formation between the incoming nucleotide and the growing primer strand. Time-resolved X-ray crystallography is used to monitor and study in real-time, at atomic resolution, the mechanism of nucleotide incorporation in crystallo. This method has been successful in studying the mechanistic details of several enzymes and we show here that it can be used to directly observe and monitor the sequential structural changes in DNA polymerase and its bound substrate DNA that are brought about by nucleotide binding to the DNA polymerase active site. iii Table of Contents List of Tables.....................................................................................................................................v List of Figures..................................................................................................................................vi List of Abbreviations...……………………………………………………………………............vii Acknowledgments..........................................................................................................................viii Chapter 1: Introduction.....................................................................................................................1 1.1 DNA Polymerases and DNA Replication.......................................................................1 1.2 DNA Polymerase Structure, Function and Organization................................................2 1.3 Mechanism of Nucleotide Incorporation and Models of Fidelity.................................10 1.4 Structural Biology of Family A DNA Polymerases......................................................15 1.5 Time-resolved X-ray crystallography for studies in enzymology.................................17 1.6 Specific aims of this work.............................................................................................21 Chapter 2: Materials and Methods..................................................................................................23 2.1 Materials........................................................................................................................23 2.2 Cloning and site-directed mutagenesis of KlenTaq DNA Polymerase.........................23 2.3 Expression and Purification of KlenTaq DNA Polymerase..........................................25 2.4 Crystallization of Dideoxy-terminated Active Ternary Complexes..............................26 2.5 X-ray Diffraction and Structure Determination............................................................27 2.6 Time-course Experiments for Nucleotide Binding and Catalysis.................................27 2.7 X-ray Diffraction of Nucleotide-soaked Ternary Complexes.......................................28 2.8 Crystallization Screening for Extension-ready Binary and Ternary Complexes..........28 2.9 Optimization of Crystallization Conditions..................................................................29 Chapter 3: Results...........................................................................................................................30 3.1 Cloning, Expression and Purification of KlenTaq DNA Polymerase...........................30 3.2 Crystallization of Dideoxy-terminated Active Ternary Complexes..............................35 3.3 Time-resolved X-ray Crystallography of Dideoxy-terminated Complexes..................40 3.4 Crystallization Screening for Extension-ready Binary and Ternary Complexes..........46 Chapter 4: Discussion......................................................................................................................50 References.......................................................................................................................................53 iv List of Tables Table I. Representative DNA polymerases and their functions from the seven families.................3 Table II. Fidelity of DNA Synthesis.................................................................................................4 v List of Figures Figure 1: Number of Replicative DNA Polymerases with Increasing Genome Complexity............2 Figure 2: Domain organization of families A and B DNA polymerase............................................6 Figure 3: Sequence of Events During Nucleotide Incorporation....................................................10 Figure 4: The Two-Metal Ion Mechanism of DNA Polymerase.....................................................14 Figure 5: Crystal Structure of the Closed Ternary Complex of KlenTaq DNA Polymerase..........16 Figure 6: Map of Bacteriophage Lambda-derived Expression Vector...........................................30 Figure 7: Purification Strategy for KlenTaq DNA Polymerase......................................................32 Figure 8: Elution Profile of KlenTaq DNA Polymerase on Heparin-Sepharose............................33 Figure 9: Elution Profile of KlenTaq DNA Polymerase on Superdex 200.....................................35 Figure 10: Crystallization of Dideoxycytidine-terminated Ternary Complex................................37 Figure 11: Metal-ion Density in Different Crystallization Conditions...........................................38 Figure 12: Crystallization of Dideoxyguanosine-terminated Ternary Complex.............................39 Figure 13: KlenTaq O-helix Movement and Interactions...............................................................42 Figure 14: Time-Course of Nucleotide Binding and O-helix Conformational Change..................44 Figure 15: Effect of Crystal Size on Reaction in crystallo..............................................................45 Figure 16: Screening for “Extension-Ready” Crystals....................................................................47 Figure 17: Optimized Conditions for “Extension-ready” dGpNHpp Ternary Complex.................48 Figure 18: Crystal Structure of “Extension-ready” dGpNpp Ternary Complex.............................49 vi List of Abbreviations DNAP - DNA-dependent DNA Polymerase KlenTaq - Klenow (Large Fragment) of Thermus aquaticus (Taq) DNA Polymerase Exo- - Exonuclease-deficient mutants (d)dNTP - (Di)deoxynucleotide Triphosphates KTQ - KlenTaq DNA Polymerase PCR - Polymerase Chain Reaction vii Acknowledgements I would like to thank Drs. Katsuhiko Murakami and J. Gregory Ferry for their support and guidance, and my committee members for their guidance and helpful suggestions, throughout my graduate studies. I would also like to thank all past and present members of the Murakami lab whose work laid the foundation for mine. I especially would like to thank Ritwika Basu and Dr. Vadim Molodtsov for the day to day interactions and previous insights into protein crystallography. I would also like to thank Drs. Hement and Neela Yennawar at the Penn State X-ray Core Facility for helpful advice in crystallography and X-ray structure determination. Thank you to the kind folks at the Macromolecular Diffraction Facility, at the Cornell High Energy Synchrotron Source for their assistance in instrumentation and X-ray data collection. I thank my family for their love, support and understanding, and their confidence in me. All of which made my graduate studies less stressful. I would especially like to thank my father for constantly encouraging me, and my eldest sister for believing in me. I would not be here without them. viii Chapter 1 Introduction 1.1 DNA Polymerases and DNA
Load more

Recommended publications
  • Biochemical Studies of Dna Polymerase Theta A
    BIOCHEMICAL STUDIES OF DNA POLYMERASE THETA A Dissertation Submitted to the Temple University Graduate Board In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY by Ahmet Y Ozdemir May 2019 Examining Committee Members: Richard T Pomerantz,PhD, Advisory Chair, Fels Institute for Cancer Research and Molecular Biology & Medical Genetics and Molecular Biochemistry Xavier Graña-Amat,PhD, Fels Institute for Cancer Research and Molecular Biology & Medical Genetics and Molecular Biochemistry Tomasz Skorski, MD, PhD, DSc, Fels Institute for Cancer Research and Molecular Biology & Microbiology and Immunology Italo Tempera, PhD, Fels Institute for Cancer Research and Molecular Biology & Microbiology and Immunology Alexander Mazin, PhD, External Member, Biochemistry & Molecular Biology, Drexel University © Copyright 2019 by Ahmet Y Ozdemir All Rights Reserved ii ABSTRACT POLQ is a unique multifunctional replication and repair gene that encodes a multidomain protein with a N-terminal superfamily 2 helicase and a C-terminal A-family polymerase. Although the function of the polymerase domain has been investigated, little is understood regarding the helicase domain. Multiple studies have reported that polymerase θ-helicase (Polθ-helicase) is unable to unwind DNA. However, it exhibits ATPase activity that is stimulated by single-stranded DNA, which presents a biochemical conundrum. In contrast to previous reports, we demonstrate that Polθ-helicase (residues 1– 894) efficiently unwinds DNA with 3'–5' polarity, including DNA with 3' or 5' overhangs, blunt- ended DNA, and replication forks. Polθ-helicase also efficiently unwinds RNA- DNA hybrids and exhibits a preference for unwinding the lagging strand at replication forks, similar to related HELQ helicase. Finally, we find that Polθ-helicase can facilitate strand displacement synthesis by Polθ-polymerase, suggesting a plausible function for the helicase domain.
    [Show full text]
  • Interactions of Dna Polymerase Theta and Ku70/80 With
    INTERACTIONS OF DNA POLYMERASE THETA AND KU70/80 WITH OXIDATIVE DNA DAMAGE by Daniel Laverty A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, MD Submitted June 2018 Abstract Oxidized abasic sites (L, C4-AP, and DOB) are formed by ionizing radiation, reactive oxygen species, and some chemotherapeutics. Like abasic sites (AP), these lesions are cytotoxic and mutagenic and must be repaired, primarily by base excision repair (BER). If left unrepaired, abasic lesions stall replication and induce mutations. Repair of oxidized abasic lesions exhibits unique challenges, however. C4-AP and DOB inactivate the lyase activity of the repair enzymes DNA polymerase β and λ. Recently, several other enzymes were shown to possess lyase activity, allowing them to excise abasic lesions. Among these are DNA polymerase θ (Pol θ) and Ku70/80 (Ku). As Pol θ promotes resistance to cancer therapies which form oxidized abasic sites, the repair and replication of these lesions by Pol θ is potentially important. Ku is a core factor for non-homologous end-joining and removes AP from double strand breaks (DSBs). The interaction of Ku with oxidized abasic sites near DSB termini is potentially important for the response to ionizing radiation, which is used as a cancer treatment. Synthetic oligonucleotides containing abasic and oxidized abasic sites were prepared, and their repair or replication by Pol θ was analyzed. Pol θ bypasses C4-AP and L with reduced efficiency relative to AP and has a strong propensity to induce frameshift mutations during bypass of AP, C4-AP, L, and the oxidized nucleobase, thymidine glycol.
    [Show full text]
  • Dna Replication in Archaea: Priming, Transferase, and Elongation Activities
    DNA REPLICATION IN ARCHAEA: PRIMING, TRANSFERASE, AND ELONGATION ACTIVITIES by Zhongfeng Zuo Bachelor degree, Beijing Technology and Business University, 1999 Submitted to the Graduate Faculty of the Kenneth P. Dietrich School of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2012 UNIVERSITY OF PITTSBURGH THE KENNETH P. DIETRICH SCHOOL OF ARTS AND SCIENCES This dissertation was presented by Zhongfeng Zuo It was defended on January 27, 2012 and approved by Stephen G. Weber, Professor, Department of Chemistry Billy Day, Professor, Department of Chemistry, Department of Pharmacy Renã A. S. Robinson, Assistant Professor, Department of Chemistry Dissertation Advisor: Michael A. Trakselis, Assistant Professor, Department of Chemistry ii DNA REPLICATION IN ARCHAEA: PRIMING, TRANSFERASE, AND ELONGATION ACTIVITIES Zhongfeng Zuo, Ph.D University of Pittsburgh, 2012 Copyright © by Zhongfeng Zuo 2012 iii DNA REPLICATION IN ARCHAEA: PRIMING, TRANSFERASE, AND ELONGATION ACTIVITIES Zhongfeng Zuo, Ph.D University of Pittsburgh, 2012 We have biochemically characterized the bacterial-like DnaG primase contained within the hyperthermophilic crenarchaeon Sulfolobus solfataricus (Sso ) and compared in vitro priming kinetics with those of the eukaryotic-like primase (PriS&L) also found in Sso . Sso DnaG exhibited metal- and temperature-dependent profiles consistent with priming at high temperatures. The distribution of primer products for Sso DnaG was discrete but highly similar to the distribution of primer products produced by the homologous Escherichia coli DnaG. The predominant primer length was 13 bases, although less abundant products of varying sizes are also present. Sso DnaG was found to bind DNA cooperatively as a dimer with a moderate dissociation constant.
    [Show full text]
  • Artificial Human Telomeres from DNA Nanocircle Templates
    Artificial human telomeres from DNA nanocircle templates Ulf M. Lindstro¨ m*, Ravi A. Chandrasekaran*, Lucian Orbai*, Sandra A. Helquist*, Gregory P. Miller*, Emin Oroudjev†, Helen G. Hansma†, and Eric T. Kool*‡ *Department of Chemistry, Stanford University, Stanford, CA 94305-5080; and †Department of Physics, University of California, Santa Barbara, CA 93106 Edited by Peter B. Dervan, California Institute of Technology, Pasadena, CA, and approved October 9, 2002 (received for review July 3, 2002) Human telomerase is a reverse-transcriptase enzyme that synthe- sizes the multikilobase repeating hexamer telomere sequence (TTAGGG)n at the ends of chromosomes. Here we describe a designed approach to mimicry of telomerase, in which synthetic DNA nanocircles act as essentially infinite catalytic templates for efficient synthesis of long telomeres by DNA polymerase enzymes. Results show that the combination of a nanocircle and a DNA polymerase gives a positive telomere-repeat amplification proto- col assay result for telomerase activity, and similar to the natural enzyme, it is inhibited by a known telomerase inhibitor. We show that artificial telomeres can be engineered on human chromo- somes by this approach. This strategy allows for the preparation of synthetic telomeres for biological and structural study of telomeres and proteins that interact with them, and it raises the possibility of telomere engineering in cells without expression of telomerase itself. Finally, the results provide direct physical support for a recently proposed rolling-circle mechanism for telomerase- independent telomere elongation. rolling-circle replication ͉ primer extension ͉ telomerase ͉ TRAP assay he telomerase enzyme synthesizes the multikilobase repeat- Ting hexamer telomere sequence (TTAGGG)n at the ends of chromosomes (1–3).
    [Show full text]
  • Family a and B DNA Polymerases in Cancer: Opportunities for Therapeutic Interventions
    biology Review Family A and B DNA Polymerases in Cancer: Opportunities for Therapeutic Interventions Vinit Shanbhag 1,2, Shrikesh Sachdev 2,3, Jacqueline A. Flores 2,3, Mukund J. Modak 4 and Kamalendra Singh 2,3,4,5,* 1 Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA; [email protected] 2 The Christopher S. Bond Life Science Center, University of Missouri, Columbia, MO 65211, USA; [email protected] (S.S.); [email protected] (J.A.F.) 3 Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65211, USA 4 Department of Microbiology, Biochemistry and Molecular Genetics 225 Warren Street, NJ 07103, USA; [email protected] 5 Department of Laboratory Medicine, Karolinska Institutet, Stockholm 141 86, Sweden * Correspondence: [email protected]; Tel.: +1-573-882-9024 Received: 13 November 2017; Accepted: 29 December 2017; Published: 2 January 2018 Abstract: DNA polymerases are essential for genome replication, DNA repair and translesion DNA synthesis (TLS). Broadly, these enzymes belong to two groups: replicative and non-replicative DNA polymerases. A considerable body of data suggests that both groups of DNA polymerases are associated with cancer. Many mutations in cancer cells are either the result of error-prone DNA synthesis by non-replicative polymerases, or the inability of replicative DNA polymerases to proofread mismatched nucleotides due to mutations in 30-50 exonuclease activity. Moreover, non-replicative, TLS-capable DNA polymerases can negatively impact cancer treatment by synthesizing DNA past lesions generated from treatments such as cisplatin, oxaliplatin, etoposide, bleomycin, and radiotherapy. Hence, the inhibition of DNA polymerases in tumor cells has the potential to enhance treatment outcomes.
    [Show full text]
  • Chloroplast and Cytosolic Triosephosphate Isomerases from Spinach: Purification, Microsequencing and Cdna Cloning of the Chloroplast Enzyme
    Plant Molecular Biology 26: 1961-1973, 1994. © 1994 KIuwer Academic Publishers. Printed in Belgium. 1961 Chloroplast and cytosolic triosephosphate isomerases from spinach: purification, microsequencing and cDNA cloning of the chloroplast enzyme Katrin Henze 1, Claus Schnarrenberger 2, Josef Kellermann 3 and William Martin 1,, l lnstitutfiir Genetik, Technische Universitiit Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, FRG (* author for correspondence); 2Institut fiir Pflanzenphysiologie und Mikrobiologie, Freie Universitiit Berlin, KOnigin-Luise-Strasse 12-16a, D-14195 Berlin, Germany; 3Max Planck Institut fiir Biochemie, Am Klopferspitz, D-82152 Martinsried, Germany Received 27 September 1994; accepted 25 October 1994 Key words: triosephosphate isomerase, isoenzyme purification, Calvin cycle enzyme, cDNA cloning, gene phylogeny Abstract Chloroplast and cytosolic triosephosphate isomerases from spinach were separated and purified to homogeneity. Bothenzymes were partially sequenced by Edman degradation. Using degenerate prim- ers designed against the amino acid sequences, a homologous probe for the chloroplast enzyme was amplified and used to isolate several full-size cDNA clones. Chloroplast triosephosphate isomerase is encoded by a single gene in spinach. Analysis of the chloroplast cDNA sequence in the context of its homologues from eukaryotes and eubacteria reveals that the gene arose through duplication of its pre- existing nuclear counterpart for the cytosolic enzyme during plant evolution. Abbreviations: TPI, triosephosphate
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2014/0079836A1 Mcdaniel (43) Pub
    US 20140079836A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0079836A1 McDaniel (43) Pub. Date: Mar. 20, 2014 (54) METHODS AND COMPOSITIONS FOR (52) U.S. Cl. ALTERING HEALTH, WELLBEING AND CPC ............... A61K 36/74 (2013.01); A61 K3I/122 LIFESPAN (2013.01) USPC ............. 424/777; 514/690: 435/375; 506/16; (71) Applicant: LifeSpan Extension, LLC, Virginia 435/6.12 Beach, VA (US) (72) Inventor: David H. McDaniel, Virginia Beach, VA (57) ABSTRACT (US) Described herein are the results of comprehensive genetic (73) Assignee: LifeSpan Extension, LLC expression and other molecular analysis p the effect s anti oxidants on biological systems, including specifically differ (21) Appl. No.: 14/084,553 ent human cells. Based on these analyses, methods and com (22) Filed: Nov. 19, 2013 positions are described for modifying or influencing the lifespan of cells, tissues, organs, and organisms. In various Related U.S. Application Data embodiments, there are provided methods for modulating the activity of the gene maintenance process in order to influence (60) Continuation of application No. 13/898.307, filed on the length and/or structural integrity of the telomere in living May 20, 2013, which is a division of application No. cells, as well as methods for modulating the rate/efficiency of 12/629,040, filed on Dec. 1, 2009, now abandoned. the cellular respiration provided by the mitochondria, mito (60) Provisional application No. 61/118,945, filed on Dec. chondrial biogenesis, and maintenance of the mitochondrial 1, 2008. membrane potential. Exemplary lifespan altering compounds include natural and synthetic antioxidants, such as plant anti Publication Classification oxidant and polyphenol compounds derived from coffee cherry, tea, berry, and so forth, including but not limited to (51) Int.
    [Show full text]
  • T7 DNA Polymerase
    Europaisches Patentamt 19 European Patent Office Office europeen des brevets © Publication number: 0 386 858 B1 12 EUROPEAN PATENT SPECIFICATION © Date of publication of patent specification © int. ci.5: C12N 9/12, // C12N15/54, 13.04.94 Bulletin 94/15 C12N15/34, C12Q1/68 © Application number: 90201139.4 @ Date of filing : 24.12.87 (54) T7 DNA polymerase. feg) Priority: 14.01.87 US 3227 © Publication number of the earlier application in 14.12.87 US 132569 accordance with Art. 76 EPC : 0 265 293 @ Date of publication of application © Proprietor : THE PRESIDENT AND FELLOWS 12.09.90 Bulletin 90/37 OF HARVARD COLLEGE 17 Quincy Street Cambridge, MA 02138 (US) © Publication of the grant of the patent : 13.04.94 Bulletin 94/15 © Inventor : Tabor, Stanley 37 Fayerweather Street @ Designated Contracting States : Cambridge, Massachusetts 02138 (US) AT BE CH DE ES FR GB GR IT LI LU NL SE Inventor : Richardson, Charles C. 78 Chestnut Hill Road Chestnut Hill, Massachusetts 02167 (US) © References cited : THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 262, no. 32, 15th November 1987,pages © Representative : Moon, Donald Keith et al 15330-15333, US; S. TABOR et al.: Selective BREWER & SON Quality House Quality Court oxidation of the exonuclease domain of bac- Chancery Lane teriophage T7 DNA polymerase" London WC2A 1HT (GB) PROC. NATL. ACAD. SCI. USA, vol. 84, July 1987, pages 4767-4771; S. TABOR et al.: DNA-sequence analysis with a modified bacteriophage T7 DNA polymerase" IDEM Virology, Vol. 95, No. 1, May 1979 CO 00 If) 00 CO 00 CO Note : Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted.
    [Show full text]
  • DNA Polymerases Drive DNA Sequencing-By-Synthesis Technologies: Both Past and Present
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE REVIEW ARTICLEprovided by Frontiers - Publisher Connector published: 24 June 2014 doi: 10.3389/fmicb.2014.00305 DNA polymerases drive DNA sequencing-by-synthesis technologies: both past and present Cheng-Yao Chen* Protein Engineering Group, Illumina, San Diego, CA, USA Edited by: Next-generation sequencing (NGS) technologies have revolutionized modern biological and Andrew F.Gardner, New England biomedical research. The engines responsible for this innovation are DNA polymerases; Biolabs, USA they catalyze the biochemical reaction for deriving template sequence information. In fact, Reviewed by: DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. Suleyman Yildirim, Walter Reed Army Institute of Research, USA Escherichia coli DNA polymerase I proteolytic (Klenow) fragment was originally utilized Andreas Marx, University of in Sanger’s dideoxy chain-terminating DNA sequencing chemistry. From these humble Konstanz, Germany beginnings followed an explosion of organism-specific, genome sequence information *Correspondence: accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic Cheng-Yao Chen, Protein Engineering bacteria/archaea were modified and tested in today’s standard capillary electrophoresis Group, Illumina, 5200 Illumina Way, San Diego, CA 92122, USA (CE) and NGS sequencing platforms.These enzymes were selected for their efficient incor- e-mail: [email protected] poration of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage φ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate- labeled nucleoside polyphosphates.
    [Show full text]
  • The Central Role of Acetyl-Coa in Plant Metabolism, As Examined Through Studies of ATP Citrate Lyase and the Bio1 Mutant of Arabidopsis Elizabeth K
    Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2004 The central role of acetyl-CoA in plant metabolism, as examined through studies of ATP citrate lyase and the bio1 mutant of Arabidopsis Elizabeth K. Winters Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Genetics Commons, Molecular Biology Commons, and the Plant Sciences Commons Recommended Citation Winters, Elizabeth K., "The ec ntral role of acetyl-CoA in plant metabolism, as examined through studies of ATP citrate lyase and the bio1 mutant of Arabidopsis " (2004). Retrospective Theses and Dissertations. 1203. https://lib.dr.iastate.edu/rtd/1203 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. The central role of acetyl-CoA in plant metabolism, as examined through studies of ATP citrate lyase and the biol mutant of Arabidopsis by Elizabeth K. Winters A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Plant Physiology Program of Study Committee: Eve Syrkin Wurtele, Co-major Professor Basil J. Nikolau, Co-major Professor Thomas Baum James T. Colbert David J. Oliver Mark Westgate Iowa State University Ames, Iowa 2004 UMI Number: 3158378 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted.
    [Show full text]
  • The Dark Side of UV-Induced DNA Lesion Repair
    G C A T T A C G G C A T genes Review The Dark Side of UV-Induced DNA Lesion Repair Wojciech Strzałka 1, Piotr Zgłobicki 1, Ewa Kowalska 1, Aneta Ba˙zant 1, Dariusz Dziga 2 and Agnieszka Katarzyna Bana´s 1,* 1 Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland; [email protected] (W.S.); [email protected] (P.Z.); [email protected] (E.K.); [email protected] (A.B.) 2 Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-12-664-6410 Received: 27 October 2020; Accepted: 29 November 2020; Published: 2 December 2020 Abstract: In their life cycle, plants are exposed to various unfavorable environmental factors including ultraviolet (UV) radiation emitted by the Sun. UV-A and UV-B, which are partially absorbed by the ozone layer, reach the surface of the Earth causing harmful effects among the others on plant genetic material. The energy of UV light is sufficient to induce mutations in DNA. Some examples of DNA damage induced by UV are pyrimidine dimers, oxidized nucleotides as well as single and double-strand breaks. When exposed to light, plants can repair major UV-induced DNA lesions, i.e., pyrimidine dimers using photoreactivation. However, this highly efficient light-dependent DNA repair system is ineffective in dim light or at night. Moreover, it is helpless when it comes to the repair of DNA lesions other than pyrimidine dimers.
    [Show full text]
  • T7 DNA Polymerase
    Product Specification Storage Temperature -25°C to -15°C TEST: SPECIFICATION: Purity (SDS-PAGE) >99% Specific Activity 13,333 U/mg Product Information SS Exonuclease Functional T7 DNA Polymerase DS Exonuclease Functional Part Number P7260F DS Endonuclease 100 U =No conversion Concentration 10,000 U/mL E.coli DNA Contamination 100 U <10 copies Unit Size 550 U Price $56.00 Quality Control Analysis: Unit Characterization Assay Product Description: Unit activity was measured using a 2-fold serial dilution T7 DNA Polymerase is the mesophilic, highly processive, method. Dilutions of enzyme were made in 1X reaction buffer replicative DNA polymerase from bacteriophage T7 that is and added to 50 µL reactions containing Calf Thymus DNA, 1X responsible for the rapid and accurate replication of the T7 DNA Polymerase Unit Cheracterization Buffer (20mM Tris- HCl, 100mM KCl, 6mM MgCl2, 0.1mM EDTA, 5mM β- virus’ genome during its infection cycle. T7 DNA Polymerase 3 is a two subunit protein, consisting of a polymerase domain mercaptoethanol), H-dTTP and 150 µM dNTPs. Reactions were (gene 5 from the T7 bacteriophage) and a processivity factor incubated 10 minutes at 37°C, plunged on ice, and analyzed (E. coli trxA gene thioredoxin) (1,2). The enzyme possesses using the method of Sambrook and Russell (Molecular Cloning, a powerful (3′→5′) nuclease activity that acts on both single v3, 2001, pp. A8.25-A8.26). and double stranded DNA and appears to be responsible for the high fidelity of this enzyme and prevents strand Protein Concentration (OD280) Measurement displacement synthesis (3,4,5). A 2.0 µL sample of enzyme was analyzed at OD280 using a Nanodrop ND-1000 spectrophotometer standardized using a Source of Protein 2.0 mg/ml BSA sample (Pierce Cat #23209) and blanked with A recombinant E.
    [Show full text]