Table S1A. Enterococcus Faecium Phage 9181 Genome Organization and Features
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Crystal Structure of the Targeting Endonuclease of the Human LINE-1 Retrotransposon
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Structure, Vol. 12, 975–986, June, 2004, 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.str.2004.04.011 Crystal Structure of the Targeting Endonuclease of the Human LINE-1 Retrotransposon Oliver Weichenrieder,1,* Kostas Repanas,1 transcriptase. Depending on the DNA integration mech- and Anastassis Perrakis* anism, two classes of retrotransposons are distin- The Netherlands Cancer Institute guished. The first class contains long terminal repeat Department of Molecular Carcinogenesis-H2 (LTR) retrotransposons and retroviruses. These retroele- Plesmanlaan 121 ments use an integrase that recognizes the LTRs of the 1066 CX Amsterdam double-stranded DNA copy. The second, much larger, The Netherlands and more ancient class includes all non-LTR retro- transposons. Those are thought to integrate via target- primed reverse transcription (TPRT), a process in which Summary reverse transcription and integration are coupled (Eick- bush and Malik, 2002; Kazazian, 2004). An endonuclease The human L1 endonuclease (L1-EN) is encoded by that is part of the same polypeptide chain as the reverse the non-LTR retrotransposon LINE-1 (L1). L1 is re- transcriptase nicks the genomic DNA and hands over sponsible for more than 1.5 million retrotransposition the resulting ribose 3Ј-hydroxyl end as a primer for re- events in the history of the human genome, contribut- verse transcription of associated template RNA (Cost ing more than a quarter to human genomic DNA (L1 et al., 2002; Luan et al., 1993). and Alu elements). L1-EN is related to the well-under- Most non-LTR retrotransposons encode an endonu- stood human DNA repair endonuclease APE1, and its clease located N-terminally of the reverse transcriptase. -
Restriction Endonucleases
Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic) 9 Restriction Endonucleases Derek Robinson, Paul R. Walsh, and Joseph A. Bonventre Background Information . 226 Which Restriction Enzymes Are Commercially Available? . 226 Why Are Some Enzymes More Expensive Than Others? . 227 What Can You Do to Reduce the Cost of Working with Restriction Enzymes? . 228 If You Could Select among Several Restriction Enzymes for Your Application, What Criteria Should You Consider to Make the Most Appropriate Choice? . 229 What Are the General Properties of Restriction Endonucleases? . 232 What Insight Is Provided by a Restriction Enzyme’s Quality Control Data? . 233 How Stable Are Restriction Enzymes? . 236 How Stable Are Diluted Restriction Enzymes? . 236 Simple Digests . 236 How Should You Set up a Simple Restriction Digest? . 236 Is It Wise to Modify the Suggested Reaction Conditions? . 237 Complex Restriction Digestions . 239 How Can a Substrate Affect the Restriction Digest? . 239 Should You Alter the Reaction Volume and DNA Concentration? . 241 Double Digests: Simultaneous or Sequential? . 242 225 Genomic Digests . 244 When Preparing Genomic DNA for Southern Blotting, How Can You Determine If Complete Digestion Has Been Obtained? . 244 What Are Your Options If You Must Create Additional Rare or Unique Restriction Sites? . 247 Troubleshooting . 255 What Can Cause a Simple Restriction Digest to Fail? . 255 The Volume of Enzyme in the Vial Appears Very Low. Did Leakage Occur during Shipment? . 259 The Enzyme Shipment Sat on the Shipping Dock for Two Days. -
Phosphate Steering by Flap Endonuclease 1 Promotes 50-flap Specificity and Incision to Prevent Genome Instability
ARTICLE Received 18 Jan 2017 | Accepted 5 May 2017 | Published 27 Jun 2017 DOI: 10.1038/ncomms15855 OPEN Phosphate steering by Flap Endonuclease 1 promotes 50-flap specificity and incision to prevent genome instability Susan E. Tsutakawa1,*, Mark J. Thompson2,*, Andrew S. Arvai3,*, Alexander J. Neil4,*, Steven J. Shaw2, Sana I. Algasaier2, Jane C. Kim4, L. David Finger2, Emma Jardine2, Victoria J.B. Gotham2, Altaf H. Sarker5, Mai Z. Her1, Fahad Rashid6, Samir M. Hamdan6, Sergei M. Mirkin4, Jane A. Grasby2 & John A. Tainer1,7 DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 50-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 50-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 50polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via ‘phosphate steering’, basic residues energetically steer an inverted ss 50-flap through a gateway over FEN1’s active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 50-flap specificity and catalysis, preventing genomic instability. 1 Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. -
Type of the Paper (Article
Supplementary Material A Proteomics Study on the Mechanism of Nutmeg-induced Hepatotoxicity Wei Xia 1, †, Zhipeng Cao 1, †, Xiaoyu Zhang 1 and Lina Gao 1,* 1 School of Forensic Medicine, China Medical University, Shenyang 110122, P. R. China; lessen- [email protected] (W.X.); [email protected] (Z.C.); [email protected] (X.Z.) † The authors contributed equally to this work. * Correspondence: [email protected] Figure S1. Table S1. Peptide fraction separation liquid chromatography elution gradient table. Time (min) Flow rate (mL/min) Mobile phase A (%) Mobile phase B (%) 0 1 97 3 10 1 95 5 30 1 80 20 48 1 60 40 50 1 50 50 53 1 30 70 54 1 0 100 1 Table 2. Liquid chromatography elution gradient table. Time (min) Flow rate (nL/min) Mobile phase A (%) Mobile phase B (%) 0 600 94 6 2 600 83 17 82 600 60 40 84 600 50 50 85 600 45 55 90 600 0 100 Table S3. The analysis parameter of Proteome Discoverer 2.2. Item Value Type of Quantification Reporter Quantification (TMT) Enzyme Trypsin Max.Missed Cleavage Sites 2 Precursor Mass Tolerance 10 ppm Fragment Mass Tolerance 0.02 Da Dynamic Modification Oxidation/+15.995 Da (M) and TMT /+229.163 Da (K,Y) N-Terminal Modification Acetyl/+42.011 Da (N-Terminal) and TMT /+229.163 Da (N-Terminal) Static Modification Carbamidomethyl/+57.021 Da (C) 2 Table S4. The DEPs between the low-dose group and the control group. Protein Gene Fold Change P value Trend mRNA H2-K1 0.380 0.010 down Glutamine synthetase 0.426 0.022 down Annexin Anxa6 0.447 0.032 down mRNA H2-D1 0.467 0.002 down Ribokinase Rbks 0.487 0.000 -
Structure and Function of Nucleases in DNA Repair: Shape, Grip and Blade of the DNA Scissors
Oncogene (2002) 21, 9022 – 9032 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Structure and function of nucleases in DNA repair: shape, grip and blade of the DNA scissors Tatsuya Nishino1 and Kosuke Morikawa*,1 1Department of Structural Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan DNA nucleases catalyze the cleavage of phosphodiester mismatched nucleotides. They also recognize the bonds. These enzymes play crucial roles in various DNA replication or recombination intermediates to facilitate repair processes, which involve DNA replication, base the following reaction steps through the cleavage of excision repair, nucleotide excision repair, mismatch DNA strands (Table 1). repair, and double strand break repair. In recent years, Nucleases can be regarded as molecular scissors, new nucleases involved in various DNA repair processes which cleave phosphodiester bonds between the sugars have been reported, including the Mus81 : Mms4 (Eme1) and the phosphate moieties of DNA. They contain complex, which functions during the meiotic phase and conserved minimal motifs, which usually consist of the Artemis : DNA-PK complex, which processes a V(D)J acidic and basic residues forming the active site. recombination intermediate. Defects of these nucleases These active site residues coordinate catalytically cause genetic instability or severe immunodeficiency. essential divalent cations, such as magnesium, Thus, structural biology on various nuclease actions is calcium, manganese or zinc, as a cofactor. However, essential for the elucidation of the molecular mechanism the requirements for actual cleavage, such as the types of complex DNA repair machinery. Three-dimensional and the numbers of metals, are very complicated, but structural information of nucleases is also rapidly are not common among the nucleases. -
Flap DNA Unwinding and Incision by the Human FAN1 Dimer
ARTICLE Received 16 Aug 2014 | Accepted 30 Oct 2014 | Published 11 Dec 2014 DOI: 10.1038/ncomms6726 Structural insights into 50 flap DNA unwinding and incision by the human FAN1 dimer Qi Zhao1,*, Xiaoyu Xue1,*, Simonne Longerich1,*, Patrick Sung1 & Yong Xiong1 Human FANCD2-associated nuclease 1 (FAN1) is a DNA structure-specific nuclease involved in the processing of DNA interstrand crosslinks (ICLs). FAN1 maintains genomic stability and prevents tissue decline in multiple organs, yet it confers ICL-induced anti-cancer drug resistance in several cancer subtypes. Here we report three crystal structures of human FAN1 in complex with a 50 flap DNA substrate, showing that two FAN1 molecules form a head-to- tail dimer to locate the lesion, orient the DNA and unwind a 50 flap for subsequent incision. Biochemical experiments further validate our model for FAN1 action, as structure-informed mutations that disrupt protein dimerization, substrate orientation or flap unwinding impair the structure-specific nuclease activity. Our work elucidates essential aspects of FAN1-DNA lesion recognition and a unique mechanism of incision. These structural insights shed light on the cellular mechanisms underlying organ degeneration protection and cancer drug resistance mediated by FAN1. 1 Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to P.S. (email: [email protected]) or to Y.X. (email: [email protected]). NATURE COMMUNICATIONS | 5:5726 | DOI: 10.1038/ncomms6726 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. -
Endonuclease VIII
Product Specifications Y9080L Rev E Product Information Product Description: E.coli Endonuclease VIII functions as both an N-glycosylase (by excising oxidative base lesions) Endonuclease VIII and an AP lyase (by subsequently cleaving the phosphodiester backbone), leaving terminal phosphates at Part Number Y9080L the 5′ and 3′ ends. (1) Damaged bases removed by Endonuclease VIII include: urea, 5, 6- dihydroxythymine, Concentration 10,000 U/mL thymine glycol, 5-hydroxy-5- methylhydanton, uracil glycol, Unit Size 10,000 U 6-hydroxy-5, 6-dihydrothymine and methyltartronylurea (2,3). Storage Temperature -25⁰C to -15⁰C Product Specifications Y9080 Specific SS E. coli DNA Assay SDS Purity DS Exonuclease Activity Exonuclease Contamination Units Tested n/a n/a 10 100 100 >99% 770,513 <1.0% <1.0% <10 copies Specification Released Released Source of Protein: An E. coli strain which carries the cloned Endonuclease VIII gene. Unit Definition: 1 unit is defined as the amount of enzyme required to cleave 1 pmol of an oligonucleotide duplex containing a single AP site in 1 hour at 37°C. Molecular weight: 29,845 Daltons Quality Control Analysis: Unit Activity is measured using a 2-fold serial dilution method. Dilutions of enzyme were prepared in Endo VIII glycerol storage solution and added to 10 µL reactions containing a FAM-labeled, 35-base, duplex oligonucleotide, containing a single Uracil. [Note: substrate was pre-treated for 2 minutes with UDG to create an abasic site] Reactions were incubated 60 minutes at 37ºC, plunged on ice, denatured with N-N-dimethylformamide and analyzed by running and exposing to short-wave UV a 15% TBE-Urea acrylamide gel. -
Leeds Thesis Template
Determining the Link Between Genome Integrity and Seed Quality Robbie Michael Gillett Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Biological Sciences School of Biology January, 2017 - ii - The candidate confirms that the work submitted is his own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. The right of Robbie M. Gillett to be identified as Author of this work has been asserted by him in accordance with the Copyright, Designs and Patents Act 1988. © 2016 The University of Leeds and Robbie Michael Gillett - iii - Acknowledgements I would like to thank my supervisor Dr Christopher West and co-supervisor Dr. Wanda Waterworth for all of their support, expertise and incredible patience and for always being available to help. I would like to thank Aaron Barrett, James Cooper, Valérie Tennant, and all my other friends at university for their help throughout my PhD. Thanks to Vince Agboh and Grace Hoysteed for their combined disjointedness and to Ashley Hines, Daniel Johnston and Darryl Ransom for being a source of entertainment for many years. I would like to thank my international Sona friends, Lindsay Hoffman and Jan Maarten ten Katen. Finally unreserved thanks to my loving parents who supported me throughout the tough times and to my Grandma and Granddad, Jean and Dennis McCarthy, who were always very vocal with their love, support and pride. -
The Microbiota-Produced N-Formyl Peptide Fmlf Promotes Obesity-Induced Glucose
Page 1 of 230 Diabetes Title: The microbiota-produced N-formyl peptide fMLF promotes obesity-induced glucose intolerance Joshua Wollam1, Matthew Riopel1, Yong-Jiang Xu1,2, Andrew M. F. Johnson1, Jachelle M. Ofrecio1, Wei Ying1, Dalila El Ouarrat1, Luisa S. Chan3, Andrew W. Han3, Nadir A. Mahmood3, Caitlin N. Ryan3, Yun Sok Lee1, Jeramie D. Watrous1,2, Mahendra D. Chordia4, Dongfeng Pan4, Mohit Jain1,2, Jerrold M. Olefsky1 * Affiliations: 1 Division of Endocrinology & Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California, USA. 2 Department of Pharmacology, University of California, San Diego, La Jolla, California, USA. 3 Second Genome, Inc., South San Francisco, California, USA. 4 Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA, USA. * Correspondence to: 858-534-2230, [email protected] Word Count: 4749 Figures: 6 Supplemental Figures: 11 Supplemental Tables: 5 1 Diabetes Publish Ahead of Print, published online April 22, 2019 Diabetes Page 2 of 230 ABSTRACT The composition of the gastrointestinal (GI) microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine (fMLF), are elevated in high fat diet (HFD)- induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon- like peptide-1 (GLP-1). Obese Fpr1-knockout (Fpr1-KO) mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota. -
Style Specifications Thesis
Structural Studies of Salvage Enzymes in Nucleotide Biosynthesis Martin Welin Faculty of Natural Resources and Agricultural Sciences Department of Molecular Biology Uppsala Doctoral thesis Swedish University of Agricultural Sciences Uppsala 2007 Acta Universitatis Agriculturae Sueciae 2007:21 ISSN 1652-6880 ISBN 978-91-576-7320-6 © 2007 Martin Welin, Uppsala Tryck: SLU Service/Repro, Uppsala 2007 Abstract Welin, M., 2007. Structural Studies of Salvage Enzymes in Nucleotide Biosynthesis. Doctoral dissertation. ISSN 1652-6880, ISBN 978-91-576-7320-6 There are two routes to produce deoxyribonucleoside triphosphates (dNTPs) precursors for DNA synthesis, the de novo and the salvage pathways. Deoxyribonucleoside kinases (dNKs) perform the initial phosphorylation of deoxyribonucleosides (dNs). Furthermore, they can act as activators for several medically important nucleoside analogs (NAs) for treatment against cancer or viral infections. Several disorders are characterized by mutations in enzymes involved in the nucleotide biosynthesis, such as Lesch-Nyhan disease that is linked to hypoxanthine guanine phosphoribosyltransferase (HPRT). In this thesis, the structures of human thymidine kinase 1 (TK1), a mycoplasmic deoxyadenosine kinase (Mm-dAK), and phosphoribosyltransferase domain containing 1 (PRTFDC1) are presented. Furthermore, a structural investigation of Drosophila melanogaster dNK (Dm-dNK) N64D mutant was carried out. The obtained structural information reveals the basis for substrate specificity for TKs and the bacterial dAKs. The TK1 revealed a structure different from other known dNK structures, containing an α/β domain similar to the RecA-F1ATPase family, and a lasso-like domain stabilized by a structural zinc. The Mm-dAK structure was similar to its human counterparts, but with some alterations in the proximity of the active site. -
Biochemical Basis for Differential Deoxyadenosine Toxicity To
Proc. Natl. Acad. Sci. USA Vol. 76, No. 5, pp. 2434-2437, May 1979 Medical Sciences Biochemical basis for differential deoxyadenosine toxicity to T and B lymphoblasts: Role for 5'-nucleotidase (deoxyadenosine kinase/deoxyadenylate kinase/immunodeficiency) ROBERT L. WORTMANN, BEVERLY S. MITCHELL, N. LAWRENCE EDWARDS, AND IRVING H. Fox Human Purine Research Center, Departments of Internal Medicine and Biological Chemistry, Clinical Research Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109 Communicated by James B. Wyngaarden, March 7, 1979 ABSTRACT Deoxyadenosine metabolism was investigated tained from Calbiochem. Erythro-9-[3-(2-hydroxynonyl)]- in cultured human cells to elucidate the biochemical basis for adenine (EHNA) was a gift from G. B. Elion of Burroughs the sensitivity of T lymphoblasts and the resistance of B lym- Wellcome (Research Triangle Park, NC). Horse serum was phoblasts to deoxyadenosine toxicity. T lymphoblasts have a 20- to 45-fold greater capacity to synthesize deoxyadenosine nu- obtained from Flow Laboratories (Rockville, MD), and Eagle's cleotides than B lymphoblasts at deoxyadenosine concentrations minimal essential medium was purchased from GIBCO. From of 50-300 ,uM. During the synthesis of dATP, T lymphoblasts Amersham/Searle, [U-14C]deoxyadenosine (505 mCi/mmol), accumulate large quantities of dADP, whereas B lymphoblasts [8-14C]hypoxanthine (52.5 mCi/mmol), and [U-14C]deoxy- do not accumu ate dADP. Enzymes affecting deoxyadenosine adenosine monophosphate (574 mCi/mmol) were purchased; nucleotide synthesis were assayed in these cells. No substantial and, from ICN, [8-14C]adenosine monophosphate (34.4 mCi/ differences were evident in activities of deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76) or mmol) was purchased (1 Ci = 3.7 X 1010 becquerels). -
Datasheet for Exonuclease V (Recbcd)
Source: An E. coli strain containing plasmids Unit Assay Conditions: 1X NEBuffer 4, 1 mM ATP Physical Purity: Purified to > 95% homogene- Exonuclease V for expressing the three subunits of E. coli with 0.15 mM sonicated duplex [3H]-DNA. ity as determined by SDS-PAGE analysis using Exonuclease V: RecB, RecC and RecD. Coomassie Blue detection. (RecBCD) Heat Inactivation: 70°C for 30 minutes. Supplied in: 100 mM NaCl, 50 mM Tris-HCl A Typical Exonuclease V Reaction: 1-800-632-7799 (pH 7.5 @ 25°C), 0.1 mM EDTA, 1 mM DTT, Quality Control Assays x µl sample DNA (~ 1 µg) [email protected] 0.1% Triton X-100 and 50% glycerol. 3 µl NEBuffer4 (10X) www.neb.com Endonuclease Activity I: Incubation of a 50 µl 3 µl 10 mM ATP M0345S 001121014101 reaction containing 100 units of Exonuclease V Reagents Supplied with Enzyme: y µl H20 (up to final volume of 30 µl) 10X NEBuffer 4, 10 mM ATP with 1 µg of φX174 RF I DNA in NEBuffer 4 and 1 µl Exonuclease V (10 units) 1 mM ATP for 4 hours at 37°C resulted in < 10% M0345S Reaction Conditions: 1X NEBuffer 4 loss in φX174 RF I DNA as determined by agarose 1. Incubate at 37°C for 30 minutes. 2. To stop reaction add EDTA to 11 mM. 1,000 units 10,000 U/ml Lot: 0011210 supplemented with 1 mM ATP. Incubate at 37°C. gel electrophoresis. 3 Heat Inactivation 70°C for 30 minutes. RECOMBINANT Store at –20°C Exp: 10/14 1X NEBuffer 4: Endonuclease Activity II: Incubation of a 50 µl 4.