Escherichia Coli
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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. -
1 Mechanistic Studies of DNA Replication, Lesion Bypass, And
Mechanistic Studies of DNA Replication, Lesion Bypass, and Editing Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Austin Tyler Raper, B.S. The Ohio State University Biochemistry Graduate Program The Ohio State University 2018 Dissertation Committee Dr. Zucai Suo, Advisor Dr. Ross Dalbey Dr. Michael Poirier Dr. Richard Swenson 1 Copyrighted by Austin Tyler Raper 2018 2 Abstract DNA acts as a molecular blueprint for life. Adenosine, cytidine, guanosine, and thymidine nucleotides serve as the building blocks of DNA and can be arranged in near- endless combinations. These unique sequences of DNA may encode genes that when expressed produce RNA, proteins, and enzymes responsible for executing diverse tasks necessary for biological existence. Accordingly, careful maintenance of the molecular integrity of DNA is paramount for the growth, development, and functioning of organisms. However, DNA is damaged upon reaction with pervasive chemicals generated by normal cellular metabolism or encountered through the environment. The resulting DNA lesions act as roadblocks to high-fidelity A- and B-family DNA polymerases responsible for replicating DNA in preparation for cell division which may lead to programmed cell death. Additionally, these lesions may fool the polymerase into making errors during DNA replication, leading to genetic mutations and cancer. Fortunately, the cell has evolved DNA damage tolerance as an emergency response to such lesions. During DNA damage tolerance, a damage-stalled high-fidelity polymerase is substituted for a specialized Y-family polymerase, capable of bypassing the offending DNA lesion, for replication to continue. -
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 -
Gene Disruption of a G4-DNA-Dependent Nuclease In
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6002-6006, June 1995 Genetics Gene disruption of a G4-DNA-dependent nuclease in yeast leads to cellular senescence and telomere shortening (guanine-quartet/KEMI/SEPI/checkpoint/meiosis) ZHIPING Liu, ARNOLD LEE, AND WALTER GILBERT Department of Molecular and Cellular Biology, The Biological Laboratories, 16 Divinity Avenue, Harvard University, Cambridge, MA 02138-2092 Contributed by Walter Gilbert, April 3, 1995 ABSTRACT The yeast gene KEMI (also named (11). A highly conserved feature is that one of the strands is SEPI/DST2/XRNI/RAR5) produces a G4-DNA-dependent very G-rich and always exists as a 3' overhang at the end of the nuclease that binds to G4 tetraplex DNA structure and cuts in chromosome. Telomeres carry out two essential functions. a single-stranded region 5' to the G4 structure. G4-DNA They protect, or stabilize, the ends of linear chromosomes, generated from yeast telomeric oligonucleotides competitively since artificially generated ends (by means of mechanical inhibits the cleavage reaction, suggesting that this enzyme sheering, x-ray irradiation, or enzymatic cleavage) are very may interact with yeast telomeres in vivo. Homozygous dele- unstable (12-14). Furthermore, they serve to circumvent the tions ofthe KEMI gene in yeast block meiosis at the pachytene incomplete DNA replication at the ends of linear chromo- stage, which is consistent with the hypothesis that G4 tetra- somes (15) by extending the G-rich strand through a de novo plex DNA may be involved in homologous chromosome pairing synthesis catalyzed by telomerase to counterbalance the se- during meiosis. We conjectured that the mitotic defects of quence loss at an end in lagging-strand replication (16, 17). -
Distinct Co-Evolution Patterns of Genes Associated to DNA Polymerase III Dnae and Polc Stefan Engelen1,2, David Vallenet2, Claudine Médigue2 and Antoine Danchin1,3*
Engelen et al. BMC Genomics 2012, 13:69 http://www.biomedcentral.com/1471-2164/13/69 RESEARCHARTICLE Open Access Distinct co-evolution patterns of genes associated to DNA polymerase III DnaE and PolC Stefan Engelen1,2, David Vallenet2, Claudine Médigue2 and Antoine Danchin1,3* Abstract Background: Bacterial genomes displaying a strong bias between the leading and the lagging strand of DNA replication encode two DNA polymerases III, DnaE and PolC, rather than a single one. Replication is a highly unsymmetrical process, and the presence of two polymerases is therefore not unexpected. Using comparative genomics, we explored whether other processes have evolved in parallel with each polymerase. Results: Extending previous in silico heuristics for the analysis of gene co-evolution, we analyzed the function of genes clustering with dnaE and polC. Clusters were highly informative. DnaE co-evolves with the ribosome, the transcription machinery, the core of intermediary metabolism enzymes. It is also connected to the energy-saving enzyme necessary for RNA degradation, polynucleotide phosphorylase. Most of the proteins of this co-evolving set belong to the persistent set in bacterial proteomes, that is fairly ubiquitously distributed. In contrast, PolC co- evolves with RNA degradation enzymes that are present only in the A+T-rich Firmicutes clade, suggesting at least two origins for the degradosome. Conclusion: DNA replication involves two machineries, DnaE and PolC. DnaE co-evolves with the core functions of bacterial life. In contrast PolC co-evolves with a set of RNA degradation enzymes that does not derive from the degradosome identified in gamma-Proteobacteria. This suggests that at least two independent RNA degradation pathways existed in the progenote community at the end of the RNA genome world. -
Glycolytic Pyruvate Kinase Moonlighting Activities in DNA Replication
Glycolytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation Steff Horemans, Matthaios Pitoulias, Alexandria Holland, Panos Soultanas, Laurent Janniere To cite this version: Steff Horemans, Matthaios Pitoulias, Alexandria Holland, Panos Soultanas, Laurent Janniere. Gly- colytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation. 2020. hal-02992157 HAL Id: hal-02992157 https://hal.archives-ouvertes.fr/hal-02992157 Preprint submitted on 10 Dec 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Glycolytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation Steff Horemans1, Matthaios Pitoulias2, Alexandria Holland2, Panos Soultanas2¶ and Laurent Janniere1¶ 1 : Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Evry, France 2 : Biodiscovery Institute, School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK Short title: PykA moonlighting activity in DNA replication Key Words: DNA replication; replication control; central carbon metabolism; glycolytic enzymes; replication enzymes; cell cycle; allosteric regulation. ¶ : Corresponding authors Laurent Janniere: [email protected] Panos Soultanas : [email protected] 1 SUMMARY Cells have evolved a metabolic control of DNA replication to respond to a wide range of nutritional conditions. -
Conversion of OX174 and Fd Single-Stranded DNA to Replicative Forms in Extracts of Escherichia Coli (Dnac, Dnad, and Dnag Gene Products/DNA Polymerase III) REED B
Proc. Nat. Acad. Sci. USA Vol. 69, No. 11, pp. 3233-3237, November 1972 Conversion of OX174 and fd Single-Stranded DNA to Replicative Forms in Extracts of Escherichia coli (dnaC, dnaD, and dnaG gene products/DNA polymerase III) REED B. WICKNER, MICHEL WRIGHT, SUE WICKNER, AND JERARD HURWITZ Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461 Communicated by Harry Eagle, August 28, 1972 ABSTRACT 4X174 and M13 (fd) single-stranded cir- MATERIALS AND METHODS cular DNAs are converted to their replicative forms by ex- tracts of E. coli pol Al cells. We find that the qX174 DNA- [a-32P]dTTP was obtained from New England Nuclear Corp. dependent reaction requires Mg++, ATP, and all four de- OX174 DNA was prepared by the method of Sinsheimer (7) oxynucleoside triphosphates, but not CTP, UTP, or GTP. or Franke and Ray (8), while fd viral DNA was prepared as This reaction also involves the products of the dnaC, dnaD, dnaE (DNA polymerase III), and dnaG genes, described (9). Pancreatic RNase was the highest grade ob- but not that of dnaF (ribonucleotide reductase). The in tainable from Worthington Biochemical Corp. It was further vitro conversion of fd single-stranded DNA to the replica- freed of possible contaminating DNase by heating a solution tive form requires all four ribonucleoside triphosphates, (2 mg/ml in 15 mM sodium citrate, pH 5) at 800 for 10 min. Mg++, and all four deoxynucleoside triphosphates. The E. protein was purified from E. coli strain reaction involves the product of gene dnaE but not those coli unwinding of genes dnaC, dnaD, dnaF, or dnaG. -
The Role of Metabolites in the Link Between DNA Replication and Central Carbon Metabolism in Escherichia Coli
G C A T T A C G G C A T genes Article The Role of Metabolites in the Link between DNA Replication and Central Carbon Metabolism in Escherichia coli Klaudyna Krause 1, Monika Maci ˛ag-Dorszy´nska 2 , Anna Wosinski 3, Lidia Gaffke 3 , Joanna Morcinek-Orłowska 1,3, Estera Rintz 3, Patrycja Biela´nska 3, Agnieszka Szalewska-Pałasz 1, Georgi Muskhelishvili 4 and Grzegorz W˛egrzyn 3,* 1 Department of Bacterial Molecular Genetics, University of Gdansk, 80-308 Gdansk, Poland; [email protected] (K.K.); [email protected] (J.M.-O.); [email protected] (A.S.-P.) 2 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 80-822 Gdansk, Poland; [email protected] 3 Department of Molecular Biology, University of Gdansk, 80-308 Gdansk, Poland; [email protected] (A.W.); lidia.gaff[email protected] (L.G.); [email protected] (E.R.); [email protected] (P.B.) 4 School of Natural Sciences, Agricultural University of Georgia, 0131 Tbilisi, Georgia; [email protected] * Correspondence: [email protected]; Tel.: +48-58-5236024 Received: 31 March 2020; Accepted: 16 April 2020; Published: 19 April 2020 Abstract: A direct link between DNA replication regulation and central carbon metabolism (CCM) has been previously demonstrated in Bacillus subtilis and Escherichia coli, as effects of certain mutations in genes coding for replication proteins could be specifically suppressed by particular mutations in genes encoding CCM enzymes. However, specific molecular mechanism(s) of this link remained unknown. -
Coordination Between Nucleotide Excision Repair And
RESEARCH ARTICLE Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria Asha Mary Joseph1, Saheli Daw1, Ismath Sadhir1,2, Anjana Badrinarayanan1* 1National Centre for Biological Sciences - Tata Institute of Fundamental Research, Bangalore, India; 2Max Planck Institute for Terrestrial Microbiology, LOEWE Centre for Synthetic Microbiology (SYNMIKRO), Marburg, Germany Abstract Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication- independent conditions. We develop an assay to follow lesion repair in non-replicating Caulobacter and observe that components of the replication machinery localize on DNA in response to damage. These localizations persist in the absence of DnaE2 or if catalytic activity of this polymerase is mutated. Single-stranded DNA gaps for SSB binding and low-fidelity polymerase-mediated synthesis are generated by nucleotide excision repair (NER), as replisome components fail to localize in the absence of NER. This mechanism of gap-filling facilitates cell cycle restoration when cells are released into replication-permissive conditions. Thus, such cross-talk (between activity of NER and specialized polymerases in subsequent gap-filling) helps preserve genome integrity and enhances survival in a replication-independent manner. *For correspondence: [email protected] Introduction Competing interests: The DNA damage is a threat to genome integrity and can lead to perturbations to processes of replica- authors declare that no tion and transcription. -
Analysis of DNA Polymerases II and III in Mutants of Escherichia Coli Thermosensitive for DNA Synthesis (Polal Mutants/Phosphocellulose Chromatography/Dnae Locus)
Proc. Nat. Acad. Sci. USA Vol. 68, No. 12, pp. 3150-3153, December 1971 Analysis of DNA Polymerases II and III in Mutants of Escherichia coli Thermosensitive for DNA Synthesis (polAl mutants/phosphocellulose chromatography/dnaE locus) MALCOLM L. GEFTER, YUKINORI HIROTA*, THOMAS KORNBERG, JAMES A. WECHSLER, AND C. BARNOUX* Department of Biological Sciences, Columbia University, New York, N.Y. 10027; and * Service de G6n6tique Cellulaire de l'Institut Pasteur, Paris Communicated by Cyrus Levinthal, October 18, 1971 ABSTRACT A series of double mutants carrying one of (5) PC79: F- his- strr malA xyl- mtlh thi- polAl sup- the thermosensitive mutations for DNA synthesis (dnaA, dnaD7 B, C, D, E, F, and G) and the polAl mutation of DeLucia and Cairns, were constructed. Enzyme activities of DNA (6) E5111: F- his- strr malA xylh mtlh arg- thi- sup- Polymerases II and III were measured in each mutant. polAl dnaE511 DNA Polymerase II activity was normal in all strains (7) E4860: F- his- str' malA xylh mtlh arg- thi- sup- tested. DNA Polymerase III activity is thermosensitive dnaE486 specifically in those strains having thermosensitive muta- tions at the dnaE locus. From these results we conclude (8) E4868: F- his- strr malA xyl- mtlh arg thi- sup- that DNA Polymerases II and III are independent en- polAl dnaE486 zymes and that DNA Polymerase III is an enzyme required (9) BT1026: H560thy-endoI- polAl dnaE for DNA replication in Escherichia coli. (10) BT1040: H560 thy endoI polAl dnaE (11) E1011: F- his- strr malA xyl- mtl- arg- thi- sup- The isolation by DeLucia and Cairns (1) of an Escherichia polAl dnaFI01 coli mutant that lacks DNA Polymerase I activity (polA1) (12) JW207: thy-rha-strrpolAl dnaF101 has prompted many investigations into the nature of the (13) NY73: leu- thy- metE rifr strr polAl dnaG3 DNA synthetic capacity of such strains. -
Coordination Between Nucleotide Excision Repair and Specialized Polymerase Dnae2 Action 2 Enables DNA Damage Survival in Non-Replicating Bacteria
bioRxiv preprint doi: https://doi.org/10.1101/2021.02.15.431208; this version posted February 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Coordination between nucleotide excision repair and specialized polymerase DnaE2 action 2 enables DNA damage survival in non-replicating bacteria 3 4 5 6 7 Asha Mary Joseph, Saheli Daw, Ismath Sadhir and Anjana Badrinarayanan* 8 9 National Centre for Biological Sciences - Tata Institute of Fundamental Research, Bellary Road, 10 Bangalore 560065, Karnataka, India, Phone: 91 80 23666547 11 *Correspondence to: [email protected] 12 13 Keywords 14 Caulobacter crescentus, DnaE2, DNA repair, error-prone polymerases, non-replicating cells, 15 nucleotide excision repair, single-cell imaging, fluorescence microscopy 16 17 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.15.431208; this version posted February 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 18 Abstract 19 Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which 20 employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models 21 suggest that activity of these polymerases is predominantly associated with ongoing replication, 22 functioning either at or behind the replication fork. -
The Escherichia Coli Polb Gene, Which Encodes DNA Polymerase II, Is Regulated by the SOS System HIROSHI IWASAKI,' ATSUO NAKATA,1 GRAHAM C
JOURNAL OF BACTERIOLOGY, Nov. 1990, p. 6268-6273 Vol. 172, No. 11 0021-9193/90/116268-06$02.00/0 Copyright © 1990, American Society for Microbiology The Escherichia coli polB Gene, Which Encodes DNA Polymerase II, Is Regulated by the SOS System HIROSHI IWASAKI,' ATSUO NAKATA,1 GRAHAM C. WALKER,2 AND HIDEO SHINAGAWA1* Department ofExperimental Chemotherapy, Research Institute for Microbial Disease, Osaka University, Suita, Osaka 565, Japan,1 and Biology Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 021392 Received 7 June 1990/Accepted 13 August 1990 The dinA (damage inducible) gene was previously identified as one of the SOS genes with no known function; it was mapped near the leuB gene, where the poiB gene encoding DNA polymerase H was also mapped. We cloned the chromosomal fragment carrying the dinA region from the ordered Escherichia coli genomic library and mapped the dinA promoter precisely on the physical map of the chromosome. The cells that harbored multicopy plasmids with the dimA region expressed very high levels of DNA polymerase activity, which was sensitive to N-ethylmaleimide, an inhibitor of DNA polymerase II. Expression of the polymerase activity encoded by the dinA locus was regulated by SOS system, and the dinA promoter was the promoter of the gene encoding the DNA polymerase. From these data we conclude that the polB gene is identical to the dinA gene and is regulated by the SOS system. The product of the polB (dinA) gene was identified as an 80-kDa protein by the maxicell method. In Escherichia coli, three DNA polymerases have been pSY343 (27), pSCH18 (10), and pRS528 (22) were used for identified (15).