A Separate Editing Exonuclease for DNA Replication
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Mutations That Separate the Functions of the Proofreading Subunit of the Escherichia Coli Replicase
G3: Genes|Genomes|Genetics Early Online, published on April 15, 2015 as doi:10.1534/g3.115.017285 Mutations that separate the functions of the proofreading subunit of the Escherichia coli replicase Zakiya Whatley*,1 and Kenneth N Kreuzer*§ *University Program in Genetics & Genomics, Duke University, Durham, NC 27705 §Department of Biochemistry, Duke University Medical Center, Durham, NC 27710 1 © The Author(s) 2013. Published by the Genetics Society of America. Running title: E. coli dnaQ separation of function mutants Keywords: DNA polymerase, epsilon subunit, linker‐scanning mutagenesis, mutation rate, SOS response Corresponding author: Kenneth N Kreuzer, Department of Biochemistry, Box 3711, Nanaline Duke Building, Research Drive, Duke University Medical Center, Durham, NC 27710 Phone: 919 684 6466 FAX: 919 684 6525 Email: [email protected] 1 Present address: Department of Biology, 300 N Washington Street, McCreary Hall, Campus Box 392, Gettysburg College, Gettysburg, PA 17325 Phone: 717 337 6160 Fax: 7171 337 6157 Email: [email protected] 2 ABSTRACT The dnaQ gene of Escherichia coli encodes the ε subunit of DNA polymerase III, which provides the 3’ 5’ exonuclease proofreading activity of the replicative polymerase. Prior studies have shown that loss of ε leads to high mutation frequency, partially constitutive SOS, and poor growth. In addition, a previous study from our lab identified dnaQ knockout mutants in a screen for mutants specifically defective in the SOS response following quinolone (nalidixic acid) treatment. To explain these results, we propose a model whereby in addition to proofreading, ε plays a distinct role in replisome disassembly and/or processing of stalled replication forks. -
DNA POLYMERASE III HOLOENZYME: Structure and Function of a Chromosomal Replicating Machine
Annu. Rev. Biochem. 1995.64:171-200 Copyright Ii) 1995 byAnnual Reviews Inc. All rights reserved DNA POLYMERASE III HOLOENZYME: Structure and Function of a Chromosomal Replicating Machine Zvi Kelman and Mike O'Donnell} Microbiology Department and Hearst Research Foundation. Cornell University Medical College. 1300York Avenue. New York. NY }0021 KEY WORDS: DNA replication. multis ubuni t complexes. protein-DNA interaction. DNA-de penden t ATPase . DNA sliding clamps CONTENTS INTRODUCTION........................................................ 172 THE HOLO EN ZYM E PARTICL E. .......................................... 173 THE CORE POLYMERASE ............................................... 175 THE � DNA SLIDING CLAM P............... ... ......... .................. 176 THE yC OMPLEX MATCHMAKER......................................... 179 Role of ATP . .... .............. ...... ......... ..... ............ ... 179 Interaction of y Complex with SSB Protein .................. ............... 181 Meclwnism of the yComplex Clamp Loader ................................ 181 Access provided by Rockefeller University on 08/07/15. For personal use only. THE 't SUBUNIT . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 182 Annu. Rev. Biochem. 1995.64:171-200. Downloaded from www.annualreviews.org AS YMMETRIC STRUC TURE OF HOLO EN ZYM E . 182 DNA PO LYM ER AS E III HOLO ENZ YME AS A REPLIC ATING MACHINE ....... 186 Exclwnge of � from yComplex to Core .................................... 186 Cycling of Holoenzyme on the LaggingStrand -
Purification and Propertiesof a DNA-Binding Protein With
Proc. Natl. Acad. Sci. USA Vol. 73, No. 7, pp. 2249-2253, July 1976 Biochemistry Purification and properties of a DNA-binding protein with characteristics expected for the Cro protein of bacteriophage X, a repressor essential for lytic growth (bacteriophage X cro gene/promoter-operator DNA) ATIS FOLKMANIS, YOSHINORI TAKEDA, JOSEF SIMUTH, GARY GUSSIN*, AND HARRISON ECHOLS Department of Molecular Biology, University of California, Berkeley, Calif. 94720, and * Department of Zoology, University of Iowa, Iowa City, Iowa 52242 Communicated by A. D. Kaiser, April 14, 1976 ABSTRACT The Cro protein specified by bacteriophage X viously (8), X DNA was labeled with P2p by growth of phage in is a repressor essential for normal lytic growth of the virus, thus low phosphate medium containing 5 ,Ci/ml of UP-labeled having a physiological role distinct from that of cI, the repressor that maintains lyso We have purified a X-specific DNA- inorganic phosphate. Phage were purified by precipitation with binding protein witheny.the requirements for synthesis and bio- polyethylene glycol and centrifugation to equilibrium in a CsCI chemical activities expected for Cro protein from studies in vivo. density gradient (2-3 times). DNA was extracted with redis- As isolated, the protein appears to be a dimer of molecular tilled phenol, the phenol was removed by extraction with ether, weight approximately 18,000 with DNA-binding properties that and the DNA was dialyzed into and stored in 10mM Tris-HCl, are very similar, but not identical, to those of the cI protein. We 0.2 mM EDTA, at pH 7.3. infer that bacteriophage X uses the same regulatory region of DNA for two different DNA-binding repressor proteins with DNA-Binding Assay. -
Pamela L. Mellon, Ph.D
Pamela L. Mellon, Ph.D. Vice-Chair for Research, Department of Reproductive Medicine Distinguished Professor, Departments of Reproductive Medicine and Neurosciences Director, Center for Reproductive Science and Medicine University of California, San Diego, School of Medicine 3A14 Leichtag Biomedical Research Building 9500 Gilman Drive, La Jolla, CA 92093-0674 (858) 534-1312, Fax (858) 534-1438, e-mail: [email protected] Departmental Web page: http://repromed.ucsd.edu/divisions/endocrinology/mellon.shtml Laboratory Web Page: http://repro.ucsd.edu/Mellon/SitePages/home.aspx ORCID 0000-0002-8856-0410 EDUCATION B.A., 1975, University of California at Santa Cruz Degrees in both Biology and Chemistry with Highest Honors Ph.D., 1979, University of California at Berkeley Department of Molecular Biology Dissertation: Two Transforming Genes and Three Replicative Genes of Avian RNA Tumor Viruses: Identification, Gene Order, and Gene Expression APPOINTMENTS Research Associate, 1975, University of California at Berkeley Department of Molecular Biology with Dr. Harrison Echols Postdoctoral Fellow with Dr. Tom Maniatis 1979-1980, California Institute of Technology, Division of Biology 1980-1984, Harvard University, Department of Biochemistry and Molecular Biology Assistant Professor 1984-1990, The Salk Institute for Biological Studies, Regulatory Biology Laboratory Assistant Adjunct Professor 1988-1991, University of California, San Diego Associate Professor 1990-1991, The Salk Institute for Biological Studies, Regulatory Biology Laboratory Associate -
Journal of Virology
JOURNAL OF VIROLOGY VOLUME 37 0 NUMBER 1 0 JANUARY 1981 EDITORIAL BOARD Robert R. Wagner, Editor-in-Chief (1982) University of Virginia School of Medicine, Charlottesville Dwight L. Anderson, Editor (1983) Haold S. Ginsberg, Editor (1984) School ofDentistry, Columbia University University of Minnesota, New York, N. Y. Minneapolis David T. Denhardt, Editor (1982) Edward M. Scolnick, Editor (1982) University of Western Ontario National Cancer Institute London, Ontario, Canada Bethesda, Md. David Baltimore (1981) Calderon Howe (1982) Dan S. Ray (1983) Amiya K. Banerjee (1982) Alice S. Huang (1981) M. E. Reichmann (1982) Kenneth I. Berns (1982) Tony Hunter (1983) Bernard E. Reilly (1983) David H. L. Bishop (1982) D. C. Kelly (1982) Wiliam S. Robinson (1983) David Botstein (1982) Thomas J. Kelly, Jr. (1982) Bernard Roizman (1982) Dennis T. Brown (1981) George Khoury (1981) Roland R. Rueckert (1982) Ahmad 1. Bukhari (1981) Jonathan A. King (1981) Norman P. Salzman (1981) Purnell Cboppin (1983) David W. Kingsbury (1982) Joseph Sambrook (1982) John M. Coffin (1983) Daniel Kolakofsky (1983) PrisciUa A. Schaffer (1981) Richard W. Compans (1982) Lloyd M. Kozboff (1982) Sondra Schlesinger (1983) Geoffrey M. Cooper (1981) Robert M. Krug (1983) June R. Scott (1983) Clive Dickson (1981) Robert A. Lazzarini 1981) Phillip A. Sharp (1982) Walter Doerfler (1983) Richard A. Lerner (1981) Aaron J. Shatkin (1982) Harrison Echols (1981) Myron Levine (1982) Saul J. Sllverstein (1982) Elvera Ehrenfeld (1983) Tomas Lindahl (1981) Lee D. Simon (1981) Robert N. Eisenman (1982) Douglas R. Lowy (1983) Kai Simons (1981) Suzanne U. Emerson (1983) Ronald B. Luftig (1981) Patrcia G. Spear (1981) Lynn Enquist (1981) Robert Martin (1981) Mark F. -
Tetrameric Uvrd Helicase Is Located at the E. Coli Replisome Due to Frequent Replication Blocks Adam J
bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432310; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Tetrameric UvrD helicase is located at the E. coli replisome due to frequent replication blocks Adam J. M Wollman1,2,3,, Aisha H. Syeda1,2, Andrew Leech4 , Colin Guy5, 6, Peter McGlynn2,6, Michelle Hawkins2 and Mark C. Leake1,2 The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors 1 Department of Physics, University of York, York YO10 5DD, United Kingdom. 2 Department of Biology, University of York, York YO10 5DD, United Kingdom. 3 Current address: Biosciences Institute, Newcastle University, NE1 7RU, United Kingdom. 4 Bioscience Technology Facility, Department of Biology, University of York, York YO10 5DD, United Kingdom 5 Current address: Covance Laboratories Ltd., Otley Road, Harrogate, HG3 1PY, United Kingdom 6 Previous address: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom * To whom correspondence should be addressed. To whom correspondence should be addressed. Tel: +44 (0)1904322697. Email: [email protected] Present Address: Departments of Physics and Biology, University of York, York YO10 5DD, United Kingdom ABSTRACT DNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli, Rep and UvrD, help replication machinery overcome blocks by removing incoming nucleoprotein complexes or aiding the re-initiation of replication. -
Duplication Mutation As an SOS Response in Escherichia Coli
Copyright 0 1989 by the Genetics Society of America Duplication Mutation as anSOS Response in Escherichia coli: Enhanced Duplication Formationby a Constitutively Activated RecA Joan Dimpfl and Harrison Echols Department of Molecular Biology, University of CaE$ornia, Berkeley, California 94720 Manuscript received March 10, 1989 Accepted for publication July 6, 1989 ABSTRACT The SOS response in Escherichia coli involves the induction of a multioperon regulatory system, which copes withthe presence of DNA lesions that interfere with DNA replication. Induction depends on activation of the RecA protein to cleave the LexA repressor of SOS operons. In addition to inducible DNA repair, the SOS system producesa large increasein the frequency of point mutations. To examine the possibility that other types of mutations are induced as partof the SOS response, we have studied the production of tandem duplications.To avoid the complications of indirect effectsof the DNA lesions, we have activated the SOS response by a constitutive mutation in the recA gene, recA730. The introduction of the recA730 mutation results in an increase in duplications in the range of tenfold or greater, as judged by two different criteria. Based on its genetic requirements, the pathway for induced duplication formation is distinct from the point mutation pathway and also differs from the major normal recombination pathway.The induction of pathways for both duplica- tions and point mutationsshows that the SOS system produces a broad mutagenic response.We have suggested previously that many typesof mutations might be inducedby severe environmental stress, thereby enhancing genetic variationin an endangered population. HE introduction of a replication-inhibitinglesion deletions, translocations) (ECHOLS198 1 ; 1982; Mc- T (by UV light or a chemical mutagen) into the DONALD1984). -
Exonucleolytic Editing by DNA Polymerase III Holoenzyme- (DNA Replication/Fidelity of Replication/Mutagenesis/Proofreading) HARRISON Echolstt, CHI Lutf, and PETER M
Proc. NatL Acad. Sci. USA Vol. 80, pp. 2189-2192, April 1983 Biochemistry Mutator strains of Escherichia coli, mutD and dnaQ, with defective exonucleolytic editing by DNA polymerase III holoenzyme- (DNA replication/fidelity of replication/mutagenesis/proofreading) HARRISON ECHOLStt, CHI Lutf, AND PETER M. J. BURGERSt§ tDeartament of Molecular Biology, University of California, Berkeley, California 94720; and tDepartment of Biochemistry, Stanford University School of Medicine, StOrd. California 94305 Communicated by I. Robert Lehman, January 17, 1983 ABSTRACT The closely linked mutD and dnaQ mutations clease. We infer that the mutD (dnaQ) gene product controls confer a vastly increased mutation rate on Escherichia coli and the editing capacity of pol III. thus might define a gene with a central role in the fidelity of DNA replication. To look for the biochemical function of the mutD gene MATERIALS AND METHODS product, we have measured the 3' -* 5' exonucleolytic editing ac- tivity of polymerase m holoenzyme from mutD5 and dnaQ49 mu- Materials. Unlabeled deoxynucleoside triphosphates and the tants. The editing activities of the mutant enzymes are defective polymers (dA)1,5oo and (dT)17 were obtained from P-L Bio- compared to wild type, as judged by two assays: (i) decreased ex- chemicals. [3H]dTTP and [3H]dTMP were purchased from New cision of a terminal mispaired base from a copolymer substrate England Nuclear and Schwarz/Mann, respectively. [a-32P]dTTP and (i) turnover of dTTP to dTMP during replication with a phage was obtained from Amersham. Polyethylenimine (PEI)-cellu- G4 DNA template. Thus, the mutD (dnaQ). gene product is likely lose plates were from Machery-Nagel and DEAE-paper (DE81) to control the editing (proofreading) capacity of polymerase HI was from Whatman. -
Q 297 Suppl USE
The following supplement accompanies the article Atlantic salmon raised with diets low in long-chain polyunsaturated n-3 fatty acids in freshwater have a Mycoplasma dominated gut microbiota at sea Yang Jin, Inga Leena Angell, Simen Rød Sandve, Lars Gustav Snipen, Yngvar Olsen, Knut Rudi* *Corresponding author: [email protected] Aquaculture Environment Interactions 11: 31–39 (2019) Table S1. Composition of high- and low LC-PUFA diets. Stage Fresh water Sea water Feed type High LC-PUFA Low LC-PUFA Fish oil Initial fish weight (g) 0.2 0.4 1 5 15 30 50 0.2 0.4 1 5 15 30 50 80 200 Feed size (mm) 0.6 0.9 1.3 1.7 2.2 2.8 3.5 0.6 0.9 1.3 1.7 2.2 2.8 3.5 3.5 4.9 North Atlantic fishmeal (%) 41 40 40 40 40 30 30 41 40 40 40 40 30 30 35 25 Plant meals (%) 46 45 45 42 40 49 48 46 45 45 42 40 49 48 39 46 Additives (%) 3.3 3.2 3.2 3.5 3.3 3.4 3.9 3.3 3.2 3.2 3.5 3.3 3.4 3.9 2.6 3.3 North Atlantic fish oil (%) 9.9 12 12 15 16 17 18 0 0 0 0 0 1.2 1.2 23 26 Linseed oil (%) 0 0 0 0 0 0 0 6.8 8.1 8.1 9.7 11 10 11 0 0 Palm oil (%) 0 0 0 0 0 0 0 3.2 3.8 3.8 5.4 5.9 5.8 5.9 0 0 Protein (%) 56 55 55 51 49 47 47 56 55 55 51 49 47 47 44 41 Fat (%) 16 18 18 21 22 22 22 16 18 18 21 22 22 22 28 31 EPA+DHA (% diet) 2.2 2.4 2.4 2.9 3.1 3.1 3.1 0.7 0.7 0.7 0.7 0.7 0.7 0.7 4 4.2 Table S2. -
Escherichia Coli Dnax Product, the 7 Subunit of DNA Polymerase
Proc. Natl. Acad. Sci. USA Vol. 84, pp. 2713-2717, May 1987 Biochemistry Escherichia coli DnaX product, the 7 subunit of DNA polymerase III, is a multifunctional protein with single-stranded DNA-dependent ATPase activity (DNA replication/dnaZX gene) SUK-HEE LEE AND JAMES R. WALKER Department of Microbiology, University of Texas, Austin, TX 78712 Communicated by Esmond E. Snell, January 7, 1987 ABSTRACT The dnaZX gene of Escherichia coli directs Germino et al. (14) have used affinity chromatography to the synthesis of two proteins, DnaZ and DnaX. These products purify a bifunctional fusion protein consisting of the initiator are confirmed as the y and X subunits of DNA polymerase HI of plasmid R6K replication fused near its C-terminal end to because antibody to a synthetic peptide present in both the ,B-galactosidase. DnaZ and DnaX proteins reacts also with the y and T subunits ofholoenzyme. To characterize biochemically the Tsubunit, for which there has been no activity assay, the dnaZX gene was METHODS fused to the 13-galactosidase gene to encode a fusion product in which the 20 C-terminal amino acids of the DnaX protein (T) Bacterial Strains and Plasmids. Strain RB791, a lacIQ L8 were replaced by fi-galactosidase lacking only 7 N-terminal derivative of strain W3110 (15), was obtained from Nina amino acids. The 185-kDa fusion protein, which retained Irwin (Harvard University). Strain M182, A(lacIPOZYA)- ,8-galactosidase activity, was overproduced to the level ofabout X74, galK, galU, rpsL (16), was obtained from Richard 5% of the soluble cellular protein by placing the gene fusion Meyer (University of Texas). -
Activity of the Hsp7o Chaperone Complex-Dnak, Dnaj, And
Proc. Natl. Acad. Sci. USA Vol. 89, pp. 12108-12111, December 1992 Biochemistry Activity of the Hsp7O chaperone complex-DnaK, DnaJ, and GrpE- in initiating phage A DNA replication by sequestering and releasing A P protein (heat shock proteins/DNA replication/protein-protein interaction) HEIDI J. HOFFMANN, SUSAN K. LYMAN, CHI Lu, MARIE-AGNES PETIT, AND HARRISON ECHOLS Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720 Contributed by Harrison Echols, September 24, 1992 ABSTRACT Initiation of DNA replication by phage A occurs efficiently with less DnaK, and the unwinding reaction requires the ordered assembly and disassembly ofa specialized is more often bidirectional, indicating a more efficient disas- nucleoprotein structure at the origin of replication. In the sembly reaction (C. Wyman, C. Vasilikiotis, D. Ang, C. disasembly pathway, a set of Escherichia coli heat shock Georgopoulos, and H.E., unpublished work). Based on a proteins termed the Hsp7O complex-DnaK, DnaJ, and variety of data, the three-protein set of heat shock proteins GrpE-act with ATP to release A P protein from the nucleo- appears likely to function typically together. DnaJ and GrpE protein complex, freeing the DnaB helicase for its DNA- markedly stimulate the ATPase activity of DnaK, the bac- unwinding reaction. To investigate the mechanism of the terial homolog ofthe eukaryotic -70-kDa heat shock protein release reaction, we have exmned the interaction between P Hsp7O (14). Moreover, the three-protein set ("Hsp7O com- and the three heat shock proteins by glycerol gradient sedi- plex") acts together in other reactions, including control of mentation and gel electrophoresis. -
Activity of the Purified Mutagenesis Proteins Umuc, Umud', and Reca
Proc. Natl. Acad. Sci. USA Vol. 89, pp. 10777-10781, November 1992 Biochemistry Activity of the purified mutagenesis proteins UmuC, UmuD', and RecA in replicative bypass of an abasic DNA lesion by DNA polymerase III (SOS respon/fdelity of DNA repfcatlon/umutatlo) MALINI RAJAGOPALANt, CHI Lut, ROGER WOODGATEtt, MIKE O'DONNELL§, MYRON F. GOODMAN$, AND HARRISON ECHOLSt tDivision of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720; 1Department of Microbiology, Cornell University Medical College, New York, NY 10021; and IDepartment of Biological Science, University of Southern California, Los Angeles, CA 90089 Contributed by Harrison Echols, August 17, 1992 ABSTRACT The introduction of a replication-inhibiting SOS mutagenesis (C. Bonner, S. Creighton and M.F.G., lesion Into the DNA ofEscherichia colU generates the Induced, unpublished work). multigene SOS response. One component of the SOS response Together, the studies noted above give clear indications is a marked increase in mutation rate, de t on RecA that SOS mutagenesis is a consequence ofreplicative bypass protein and the induced muta p us UmuC and of DNA lesions mediated by a damage-localized nucleopro- UmuD. A variety of previous indirect ap es have Indi- tein complex involving RecA, UmuC-UmuD', and pol III-a cated that SOS mutagenesi results from replicative bypass of "mutasome" (14, 17). However, direct evidence for such a the DNA lesion by DNA polymerase (pol ) me in pathway has been lacking in the absence of a defined bio- a reaction mediated by RecA, UmuC, and a prcsd form of chemical system. In the work reported here, we have used UmuD termed UmuD'.