DOCSLIB.ORG

III of Escherichia Coli: Characterization of a Hole Mutant and Comparison with a Dnaq (6-Subunit) Mutant STEVEN C

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
III of Escherichia Coli: Characterization of a Hole Mutant and Comparison with a Dnaq (6-Subunit) Mutant STEVEN C JOURNAL OF BACTERIOLOGY, Feb. 1994, p. 815-821 Vol. 176, No. 3 0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology holE, the Gene Coding for the 0 Subunit of DNA Polymerase III of Escherichia coli: Characterization of a holE Mutant and Comparison with a dnaQ (6-Subunit) Mutant STEVEN C. SLATER,'t MIRIAM R. LIFSICS,1 MIKE O'DONNELL,2'3 AND RUSSELL MAURER'* Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4960,1 and Department of Microbiology, Cornell University Medical College,2 and Howard Hughes Medical Institute,3 New York, New York 10021 Downloaded from Received 4 October 1993/Accepted 2 December 1993 DNA polymerase III holoenzyme is a multiprotein complex responsible for the bulk of chromosomal replication in Escherichia coli and Salmonella typhimurium. The catalytic core of the holoenzyme is an arO heterotrimer that incorporates both a polymerase subunit ((; dnaE) and a proofreading subunit (£; dnaQ). The role of 0 is unknown. Here, we describe a null mutation ofholE, the gene for 0. A strain carrying this mutation was fully viable and displayed no mutant phenotype. In contrast, a dnaQ null mutant exhibited poor growth, chronic SOS induction, and an elevated spontaneous mutation rate, like dnaQ null mutants of S. typhimurium described previously. The poor growth was suppressible by a mutation affecting a which was identical to a http://jb.asm.org/ suppressor mutation identified in S. typhimurium. A double mutant null for both holE and dnaQ was indistinguishable from the dnaQ single mutant. These results show that the 0 subunit is dispensable in both dnaQ+ and mutant dnaQ backgrounds, and that the phenotype of e mutants cannot be explained on the basis of interference with 0 function. A goal of research on DNA polymerase III (Pol III) £, but not to a, and has no detectable effect on the polymerase holoenzyme is the elucidation of the precise role of each of its activity of a or an as complex. The only activity so far 10 or more distinct subunits. Research thus far has identified identified for 0 is a threefold stimulatory effect on the exonu- on August 7, 2015 by Rockefeller University the a subunit as the polymerase proper (22), the E subunit as clease activity of £ on a mispaired primer terminus (40). the proofreading exonuclease (34), and the 3 subunit as a Here, we report the construction of the first 0 (holE) doughnut-shaped clamp that topologically tethers the poly- mutation and describe its properties, both as a single mutation merase to the DNA and makes it highly processive (15, 29, 41). and in combination with a null mutation in dnaQ. In addition, the T subunit dimerizes the holoenzyme to allow coordinated leading- and lagging-strand synthesis by twin polymerases (27, 39). Most of the remaining subunits are MATERIALS AND METHODS involved in controlling the assembly of ,B with the template- primer, but the individual subunit roles in this function are not Bacterial strains and media. Bacterial strains and plasmids yet clear. are listed in Table 1. Phage Plvir was used for transductions. At least some of the subunits of the holoenzyme are Bacteria were generally maintained at 37°C on Luria-Bertani multifunctional. s, for example, affects the rate, processivity, (LB) medium (36). Sucrose plates were NaCl-free LB plus 5% and thermal stability of the enzyme, not just its fidelity (22, 23, sucrose (3). Antibiotics were used at the following concentra- 38). Null mutants affected in s, obtained in Salmonella typhi- tions (in micrograms per milliliter): ampicillin, 100; kanamycin, murium, exhibit both an elevated mutation rate and a severe 50; tetracycline, 25; chloramphenicol, 15; rifampin, 100; nali- growth defect (16). These phenotypes are experimentally dixate, 50. distinct because the growth defect can be specifically sup- Enzymes and reagents. Restriction enzymes were from New pressed by mutations affecting a (16, 17). Genetic and bio- England Biolabs, United States Biochemical, or Boehringer chemical analysis suggests that the s mutants suffer from Mannheim. Sequenase was from United States Biochemical. insufficient polymerization capacity and that the suppressors GeneScreen Plus hybridization membrane was from Dupont. increase this capacity (16, 19). Radiolabelled nucleotides were from Amersham. Western To date, the role of the 0 subunit has remained elusive. Early blots (immunoblots) were performed with a Tropix lumines- efforts to define its role biochemically were frustrated by the cent labelling kit. SeaKem agarose was from FMC. Constitu- scarcity of the protein and by the difficulty of preparing other ents of bacteriological media were from Difco. Other reagents core subunits free of 0. Conversely, the lack of 0 mutants has were from Sigma. prevented genetic analysis. Recently, however, the gene for 0 holE constructs. The holE disruption mutants were initially has been identified by reverse genetics (5, 40), making it constructed by using plasmid pUC18-0, which contains holE on possible to overproduce and purify 0 and begin its character- a 2.7-kb EcoRV fragment from Kohara X clone 336 (40). The ization. Initial biochemical analysis shows that 0 binds tightly to plasmid contains a unique EagI site 65 bp downstream of the holE initiation codon. It also contains three NruI sites, all within the insert. Site 1 is located approximately 1.7 kb * Corresponding author. Phone: 216-368-6021. Fax: 216-368-3055. upstream of holE, very near the insert-vector junction. Site 2 is Electronic mail address: [email protected]. within holE, 156 bp downstream of the holE initiation codon. t Present address: Department of Biochemistry and Molecular Site 3 is approximately 270 bp downstream of the holE Biology, Harvard University, Cambridge, MA 02138. termination codon. The antibiotic marker used for disruption 815 816 SLATER ET AL. J. BACrERIOL. TABLE 1. E. coli strains and plasmids Species and strain or plasmid Relevant characteristic(s) Source or reference E. colia RM990 A(lac proAB) thi rpsL supE endA sbcBIS hsdR4/F' traD36 25 proAB+ laclq A(lacZMS) RM1094 HfrH 25 RM1734 (=MG1655) F- wild type 1 RM2541 E. coli C-1 (susceptible to phage 4X174) E. coli Genetic Stock Center RM3979 RM1094 dnaQ903::tet Allele exchange (pFF596) RM3980 RM1734 dnaQ903::tet spq-2 P1 * RM3979 and spontaneous suppressor RM4006 RM1734 malE::TnS lexA3 (Ind-) This laboratory RM4021 RM1094 holE201::cat Allele exchange (pFF608) RM4029 (=CAG18580) RM1734 zae-3095::TnlOkan 35 Downloaded from RM4030 RM1734 holE201::cat P1 RM4021 RM4074 RM1094 holE202::cat Allele exchange (pFF622) RM4193 RM1734 holE202::cat Pl - RM4074 RM4194 RM1734 dnaQ903::tet spq-2 holE202::cat RM3980 + P1 RM4193 RM4196 RM1734 zae-3095::TnlOkan spq-2 RM3980 + P1 * RM4029 RM4221 (=W1485) F+ wild type 1 Plasmid pBIP3 Allele replacement vector 36 pFF441 mucA'B 37 http://jb.asm.org/ pFF498 mucA' 37 pFF499 mucB 37 pFF588 dnaQ This laboratory pFF589 cat This laboratory pFF590 tet This laboratory pFF596 pBIP3 derivative carrying dnaQ903::tet This work pFF608 pBIP3 derivative carrying holE201::cat This work pFF622 pBIP3 derivative carrying holE202::cat This work pUC18-0 holE+ 40 on August 7, 2015 by Rockefeller University a E. coli K-12 except as indicated. (cat, chloramphenicol resistance) was derived from pFF589, from one of the transformants. This plasmid contains the which is pBluescript IISK+ (Stratagene) carrying cat on a expected replacement of the dnaQ BssHII-MluI fragment with BamHI fragment of pJD12 (11). cat was excised from pFF589 tet. This deletion-insertion mutation, which we designated by digestion with EagI and SmaI. Plasmid pUC18-0 was dnaQ903, leaves the rnh promoter intact (28) and is very similar partially digested with NruI, and linearized plasmid was gel to the dnaQ200 allele previously constructed in S. typhimurium purified. This DNA was then digested to completion with EagI (16). The entire insert from pFF593 was cloned into the and ligated to the cat cassette. Escherichia coli RM990 was polylinker of pBIP3 (36), and the resulting plasmid (pFF596) transformed with the ligation products (6). As expected, three was used to replace the E. coli wild-type allele as described classes of recombinants were produced, each one deleting below. DNA from the EagI site to a different NruI site. In pFF605, cat Replacement ofchromosomal dnaQ and holE with disrupted replaced DNA from the EagI site through NruI site 3, while in aileles. Exchange of the disrupted alleles for the chromosomal pFF617 the replacement interval ended at NruI site 2. These wild-type alleles of strain RM1094 was performed by two-step alleles were designated hoIE201 and holE202, respectively. plasmid integration-excision using the method of Slater and Each disrupted allele was cloned into the polylinker of plasmid Maurer (36). The excision step was carried out in the presence pBIP3 (36), and the resulting plasmids (pFF608 carrying of tetracycline (for the dnaQ replacement) or chloramphenicol holE201 and pFF622 carrying holE202) were used to replace (for the holE replacement) to select for the excision product the E. coli wild-type allele as described below. preserving the disrupted allele. dnaQ constructs. Plasmid pFF588 is a derivative of pBlue- Mutation frequency analysis. The number of mutation script II SK+ that carries E. coli dnaQ on a 1.6-kb EcoRI events per culture was estimated from the number of mutants fragment subcloned from XRM406, -a dnaQ+ clone in our observed in at least 10 independent cultures of each strain, by collection. Plasmid pFF588 was digested to completion with the method of maximal likelihood as described previously (16, BssHII and MluI, deleting most of the 3' half of dnaQ. The 18). The standard deviation of this estimate was calculated vector fragment containing the remainder of dnaQ was end from Fig. 3 of Lea and Coulson (18). The estimate and its filled (31) and gel purified.
Load more

Recommended publications
  • 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.
    [Show full text]
  • 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
    [Show full text]
  • 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).
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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).
    [Show full text]
  • 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.
    [Show full text]
  • Using Genomics to Study Susceptibility and Resistance To
    Integrating Undergraduates into Educationally Meaningful Research Programs and Courses Jeffrey H. Miller University of California, Los Angeles CA Challenge Involve Students in Research Goals 1. Transmit the excitement of tackling the unknown 2. Have rapid rewards instead of long apprenticeship 3. Present an intellectual process 4. Student has ownership of a project and results, not a “cog in the wheel” 5. Understand the practical importance of the relevant area of research Requirements • Commitment from mentor • Commitment from student • Projects that allow goals to be achieved Environmental Samples Collected and Source: Strain Species Color Source CB001 E. coli strain Pinkish red Lab contaminant CB002 Staphylococcus strain White Amie Fong finger CB003 Bacillus cereus Salmon Soil CB004 Bacillus cereus Pale yellow Soil CB005 Bacillus cereus Yellow orange Soil CB006 Bacillus cereus Orange Soil CB007 Bacillus cereus Yellow Amie Fong finger CB008 Bacillus cereus Neon red Amie Fong hair CB009 E. fergusonii (potentially) Bloody red/orange South campus dumpster CB010 Bacillus cereus Baby pink UCLA campus CB011 Staphylococcus hominis Tan Arrowhead water jug from CHS CB012 Bacillus cereus Bright red Orthopaedic hospital bathroom handle CB013 Staphylococcus strain White #2 Orthopaedic hospital bathroom handle 8 12 11 1 13 7 10 2 M. Luteus 6 3 recC/tolC 9 5 4 Bacterial Art - Sydney Brenner WT 1. tolC 2. tolC recC Strain A=WT (yellow) starting mixture 1:20 Strain A: Strain B Strain B=Drug sensitive purple mutant (purple) growth without drug growth
    [Show full text]
  • 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).
    [Show full text]