DOCSLIB.ORG

T5 DNA Polymerase: Structural-Functional Relationships to Other DNA Polymerases (DNA Polymerase I/Proofreading/Processivity/Evolution) MARK C

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
T5 DNA Polymerase: Structural-Functional Relationships to Other DNA Polymerases (DNA Polymerase I/Proofreading/Processivity/Evolution) MARK C Proc. Nati. Acad. Sci. USA Vol. 86, pp. 4465-4469, June 1989 Biochemistry T5 DNA polymerase: Structural-functional relationships to other DNA polymerases (DNA polymerase I/proofreading/processivity/evolution) MARK C. LEAVITT AND JUNETSU ITO Department of Microbiology and Immunology, University of Arizona Health Sciences Center, Tucson, AZ 85724 Communicated by Lester 0. Krampitz, April 10, 1989 (receivedfor review February 1, 1989) ABSTRACT T5 DNA polymerase, a highly processive sin- proceed through double-stranded regions in template sec- gle-polypeptide enzyme, has been analyzed for its primary ondary structures or supercoiled plasmid templates. structural features. The amino acid sequence of T5 DNA We present here the DNA sequence of the T5 DNA polymerase has a high degree of homology with that of DNA polymerase gene* and the deduced amino acid sequence ofits polymerase I from Escherichia coli and retains many of the product. Comparisons of the primary structure of this en- amino acid residues that have been implicated in the 3' -* 5' zyme with other DNA polymerases suggest differences that exonuclease and DNA polymerase activities of that enzyme. may account for the high processivity of this enzyme. We Alignment with sequences of polymerase I and T7 DNA poly- also demonstrate the conservation of residues thought to be merase was used to identify regions possibly involved in the intimately involved in 3' -* 5' exonuclease and polymerase high processivity of this enzyme. Further, amino acid sequence activities. Finally, two amino acid sequence segments, which comparisons ofT5 DNA polymerase with a large group ofDNA may be involved in the 3' -*5' exonuclease function of these polymerases previously shown to exhibit little similarity to enzymes, appear to be highly conserved among a wide polymerase I indicate certain sequence segments are shared variety of DNA polymerases. among distantly related DNA polymerases. These shared re- gions have been implicated in the 3' -5' exonuclease function MATERIALS AND METHODS of I, which suggests that the proofreading domains polymerase DNA Sequencing. T5 was obtained from R. Fujimura (Oak of all these enzymes may be evolutionarily related. Ridge National Laboratory, Oak Ridge, TN). Phage T5 DNA was isolated from lysates of wild-type phage. T5 Bal I Bacteriophage T5 produces its own DNA polymerase that is fragments 11 and 12 were cloned into M13mp9 or -mpl9 (6) essential for phage DNA replication (1). This DNA polymer- and a nested set of deletions were created using the method ase is unusual in that it is highly processive; it extensively of Dale (7). Both strands were sequenced using either Se- elongates a primer before disassociating from the primer quenase (United States Biochemical) or the method of template and is capable of strand displacement. T5 DNA Maxam and Gilbert (8). A phage DNA fragment that included polymerase is more processive than any other single- the Bal I fragment 11-12junction was sequenced to eliminate polypeptide DNA polymerase on comparable templates (2, the possibility of a small intervening Bal I fragment at this 3). Similarly, its 3' -* 5' exonuclease or proofreading activity location. will processively hydrolyze hundreds of nucleotides before Amino Acid Sequence Comparisons. Protein similarity disassociation using either double- or single-stranded DNA searches of the National Biomedical Research Foundation substrates (4). The structural characteristics that confer high protein sequence library were performed (Release 16, March processivity upon polymerases are currently not understood, 1988) using FASTA (9). Amino acid sequence comparisons or perhaps because other well-studied DNA polymerases re- alignments were accomplished using LFASTA (9) or BESTFIT quire additional proteins to become processive. In contrast, or GAP (from the University ofWisconsin Genetics Computer processivity is an intrinsic property of T5 DNA polymerase Group). and therefore it is an appropriate subject for investigation in the area of DNA replication. Furthermore, this polymerase is also rare in its ability to RESULTS utilize nicked circular duplex DNA as a template and can DNA and Amino Acid Sequences of T5 DNA Polymerase. unwind the parental DNA strand from its template as it The physical location of the T5 DNA polymerase gene has synthesizes the new DNA strand from the 3'-OH end of the been identified (10) by restriction fragment rescue of poly- nick. The only other DNA polymerase capable of using a merase amber mutants, as being within the region of Bal I nicked template or of strand displacement in the absence of restriction fragments 11 and 12 at 58.3-61.3% of the distance other protein factors is Escherichia coli DNA polymerase I from the left end of the genome. DNA sequence analysis of (Pol I) or its large (Klenow) proteolysis product (4, 5). The Bal I fragments 11 and 12 reveals an open reading frame of dual properties of high processivity and strand displacement 2487 nucleotides and suitable ribosome binding site that may make T5 DNA polymerase well suited for use in dide- would code for a protein of 94.3 kDa (Fig. 1). This is in rough oxynucleotide DNA sequencing. The high processivity of agreement with the predicted molecular mass of T5 DNA modified T7 DNA polymerase-thioredoxin complex, known polymerase (96 kDa), as estimated by SDS/polyacrylamide commercially as Sequenase, has made it very popular for use gel electrophoresis (13), and is consistent with the direction in DNA sequencing projects. Additionally, the strand-dis- of transcription of the T5 DNA polymerase gene, as deter- placement ability of T5 DNA polymerase may enable it to mined by Schneider et al. (12). The publication costs of this article were defrayed in part by page charge Abbreviation: Pol l, DNA polymerase I. payment. This article must therefore be hereby marked "advertisement" *The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M24354). 4465 Downloaded by guest on September 26, 2021 4466 Biochemistry: Leavitt and Ito Proc. Natl. Acad. Sci. USA 86 (1989) BalI map of T5 genome (121 kb) 1 4 9 10 7 5 6 1211 8 2 13 3 I I I I I I I I I . 0.5 kb A polymerase gene rTMi BalIji BalI HpaI EcoRI BalI SmaI RBS -35 -10 -1 ATCTATICCATATCIW~ ~ ~ ~ ~ ~ ~ ~ ~ a ATA7AGATl~fTATrAG 120 M Y S I C V T R S C PPVV V CCSS K KKHH I TIT I C TPT P ENNPP F DPD PN DYD Y D FVIF V I LYL V C 40 aMYS=AI~CVCTRSC 240 A E P FL Y F A G K K G I G D Y T G K R V E Y N G YA N W I A S I S P A Q L H F 80 360 K P E M K P V F D A T V E N I H D I I N G R E K I A KA G D Y R P I'T D P D E A 120 480 E E Y I K M V Y N M V I G P V A F D S E T S A L Y C R D G Y L L C V S I S H Q E 160 600 Y Q C V Y I D S D C L T E V A V Y Y L Q K I L D S E N H T I V F H N LK F D M H 200 TATMCTACC llNCGAAIClAMOCACAT_A_A A 720 F Y K Y HL G L T F D K A H K E R R L H D T M L Q H Y L D E R R G T H G L K S 240 840 L A M K Y TD M G D Y D F E L D K F K D D Y C K A H K I K K E DF T Y D L I P F 280 . Ba1I 960 D I M W P Y A AK D T D A T I R L H N F F L P K I E K N E K L CS L Y Y D V L M 320 1080 P G C V F L Q R V E D R G V P I S I D R L K E A Q Y Q L T H N L N K A R E K L Y 360 1200 T Y P E V K Q L E Q D Q N E A F N P N S V K Q L R V L L F D Y V G L T P T G K L 400 1340 T D T G A D S T D A E A L N E L A T Q H P I A K T L L E I R K L T K L I S T Y V 440 1440 E K I L L S I D A D G C I R T G FH E H M T T S G R L S SSS K L N L Q Q L P R 480 1560 D E S I I K G C V A P P G Y R V I A W D L T T A E V Y Y A A V L S G D R N M Q 520 1680 Q V F I N M R N E P D K Y P D F H S N I A H M V F K L Q C E P R D V K K L F P A 560 1800 L R Q A AKA I T F G I L Y G S G P A KV A H S V N E A L L E Q AA K T G E P F 600 1920 V E C T V A D A K E Y I E T Y F G Q F P Q L K R W I D K C If D Q I K NH G F I Y 640 2040 S H F G R K R R L H N I H S E D R G V Q G E E I R S G F N A I I Q S A S S D S L 680 CmrA 1111 2160 L L G A V D A D NE I I S L G L E Q E M K I V M L V H D S V V A I V R E D L I D 720 CAATACMSAAMAAA~cC~r~trAATA~h~AGAAAG~c1'iarA'c1AcUI X 2280 GIS I P GC P I G I D S D S E A G G S R D Y S C 760 Q Y N E I L I R N I Q K D R EcoRI.
Load more

Recommended publications
  • Copyright by Young-Sam Lee 2010
    Copyright by Young-Sam Lee 2010 The Dissertation Committee for Young-Sam Lee Certifies that this is the approved version of the following dissertation: Structural and Functional Studies of the Human Mitochondrial DNA Polymerase Committee: Whitney Yin, Supervisor Ian Molineux Kenneth Johnson Tanya Paull Jon Robertus Structural and Functional Studies of the Human Mitochondrial DNA Polymerase by Young-Sam Lee, B.S, M.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin August, 2010 Dedication For my wife, In-Sook Jung. Acknowledgements I would like to appreciate Dr. Whitney Yin for giving me chance to working in her lab and mentoring me through my graduate program. Not only the scientific insights, also the warmness that she gave me and my family encouraged me to pursue my Ph. D. degree in the foreign country. I also would like to thank “a guru of molecular biology” Dr. Ian Molineux and “a guru of enzyme kinetics” Dr. Kenneth Johnson. Without their critical advice, I would not be accomplished my publication. I hope to be a respectable expert in my research field like them. I also should remember friendship and generosity given by many current and former Yin lab members: Hey-Ryung Chang, Qingchao “Eric” Meng, Xu Yang, Jeff Knight, Dr. Michio Matsunaga, Dr. He “River” Quan, Taewung Lee, Xin “Ella” Wang, Jamila Momand, and Max Shay. Most of all, I really appreciate my parents for their endless love and support, and my wife, In-Sook Jung, and my son, Jason Seung-Hyeon Lee who always stand by me with patients during my graduate carrier.
    [Show full text]
  • Klenow Fragment, #EP0054
    Description Klenow Fragment is the Large Fragment of DNA Polymerase I, E.coli . It exhibits 5' →3' polymerase activity and 3' →5' exonuclease (proofreading) activity, but lacks 5' →3' exonuclease activity of DNA Polymerase I. PRODUCT INFORMATION Applications Klenow Fragment • DNA blunting by fill-in of 5’-overhangs or removal of 3‘-overhangs. (1), see protocols on back page. • Random-primed DNA labeling (2-4). #EP0054 300 U • Labeling by fill-in 5 ’-overhangs of dsDNA. Lot: _ Expiry Date: _ • DNA sequencing by the Sanger method (5). • Site-specific mutagenesis of DNA with synthetic oligonucleotides (6). Concentration: 2 U/µL • Second strand synthesis of cDNA (7). Source Supplied with: 1 mL of 10X Reaction Buffer E.coli cells with a cloned fragment of the polA gene. Molecular Weight 68 kDa monomer. Store at -20 °C Definition of Activity Unit One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 min at 37°C. Enzyme activity is assayed in the following mixture: 50 mM Tris-HCl (pH 8.0 at 25°C), 5 mM MgCl 2, 1 mM DTT, In total 2 vials. 0.033 mM dNTP, 0.4 M Bq/mL [3H]-dTTP and 62.5 µg/mL activated salmon milt DNA. www.thermoscientific.com/onebio Rev.9 V Storage Buffer CERTIFICATE OF ANALYSIS The enzyme is supplied in: 25 mM Tris-HCl (pH 7.5), Endodeoxyribonuclease Assay 0.1 mM EDTA, 1 mM DTT and 50% (v/v) glycerol. 10X Reaction Buffer No conversion of covalently closed circular DNA to nicked DNA was detected after incubation of 20 units of Klenow 500 mM Tris-HCl (pH 8.0 at 25°C), 50 mM MgCl 2, 10 mM DTT.
    [Show full text]
  • PURIFIED THERMOSTABLE NUCLEIC ACID POLYMERASE ENZYME from $I(TERMOTOGA MARITIMA)
    Europäisches Patentamt *EP000544789B1* (19) European Patent Office Office européen des brevets (11) EP 0 544 789 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.7: C12N 15/54, C12N 9/12 of the grant of the patent: 05.03.2003 Bulletin 2003/10 (86) International application number: PCT/US91/05753 (21) Application number: 91915802.2 (87) International publication number: (22) Date of filing: 13.08.1991 WO 92/003556 (05.03.1992 Gazette 1992/06) (54) PURIFIED THERMOSTABLE NUCLEIC ACID POLYMERASE ENZYME FROM $i(TERMOTOGA MARITIMA) GEREINIGTES THERMOSTABILES NUKLEINSÄURE-POLYMERASEENZYM AUS THERMOTOGA MARITIMA ENZYME D’ACIDE NUCLEIQUE THERMOSTABLE PURIFIEE PROVENANT DE L’EUBACTERIE $i(THERMOTOGA MARITIMA) (84) Designated Contracting States: (74) Representative: Poredda, Andreas et al AT BE CH DE DK ES FR GB GR IT LI LU NL SE Roche Diagnostics GmbH, Patentabteilung, (30) Priority: 13.08.1990 US 567244 Sandhofer Strasse 116 68305 Mannheim (DE) (43) Date of publication of application: 09.06.1993 Bulletin 1993/23 (56) References cited: • CHEMICAL ABSTRACTS, vol. 105, no. 5, 04 (73) Proprietor: F. HOFFMANN-LA ROCHE AG August 1986, Columbus, OH (US); R. HUBER et 4002 Basel (CH) al., p. 386, AN 38901u • JOURNAL OF BIOLOGICAL CHEMISTRY, vol. (72) Inventors: 264, no. 11, 15 April 1989, American Society for • GELFAND, David, H. Biochemistry & Molecular Biology Inc., Oakland, CA 94611 (US) Baltimore, MD (US); F.C. LAWYER et al., pp. • LAWYER, Frances, C. 6427-6437 Oakland, CA 94611 (US) • STOFFEL, Susanne El Cerrito, CA 94530 (US) Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted.
    [Show full text]
  • Glucokinase Regulatory Protein Is Essential for the Proper Subcellular Localisation of Liver Glucokinase
    FEBS Letters 456 (1999) 332^338 FEBS 22420 Glucokinase regulatory protein is essential for the proper subcellular localisation of liver glucokinase Nu¨ria de la Iglesiaa, Maria Veiga-da-Cunhab, Emile Van Schaftingenb, Joan J. Guinovarta, Juan C. Ferrera;* aDepartament de Bioqu|¨mica i Biologia Molecular, Universitat de Barcelona, Mart|¨ i Franque©s, 1, E-08028 Barcelona, Spain bLaboratory of Physiological Chemistry, Christian de Duve Institute of Cellular Pathology and Universite¨ Catholique de Louvain, B-1200 Brussels, Belgium Received in revised form 24 June 1999 pressed by fructose 1-phosphate, both of which bind to Abstract Glucokinase (GK), a key enzyme in the glucose homeostatic responses of the liver, changes its intracellular GKRP and modify its a¤nity for GK [4]. This 68 kDa protein localisation depending on the metabolic status of the cell. Rat is only found in the livers of species that express GK and liver GK and Xenopus laevis GK, fused to the green fluorescent although there is some evidence for its presence in pancreatic protein (GFP), concentrated in the nucleus of cultured rat tissue [5,6], a direct demonstration is still not available. hepatocytes at low glucose and translocated to the cytoplasm at In in vitro assays, rat GKRP can inhibit human pancreatic high glucose. Three mutant forms of Xenopus GK with reduced GK, which shows a high degree of identity to the rat liver affinity for GK regulatory protein (GKRP) did not concentrate isoform [7], as well as Xenopus laevis liver GK [8], a more in the hepatocyte nuclei, even at low glucose. In COS-1 and distantly related protein.
    [Show full text]
  • Klenow Fragment Is a Mesophilic DNA Polymerase Derived from the E.Coli Polymerase I DNA- Klenow Fragment Dependent Repair Enzyme
    Product Specifications P7060L Rev C Product Information Product Description: Klenow Fragment is a mesophilic DNA polymerase derived from the E.coli Polymerase I DNA- Klenow Fragment dependent repair enzyme. The enzyme exhibits DNA synthesis and proofreading (3′→5′) nuclease activities, and, Part Number P7060L in the absence of the holoenzyme’s (5′→3′) nuclease domain, displays a moderate strand displacement activity Concentration 5,000 U/mL during DNA synthesis. The protein is expressed as a Unit Size 2,500 U truncated product of the E.coli PolA gene. Storage Temperature -25⁰C to -15⁰C Product Specifications P7060 Specific SS DS E. coli DNA Assay SDS Purity DS Exonuclease Activity Exonuclease Endonuclease Contamination Units Tested n/a n/a 50 50 50 50 Specification >99% 5,000 U/mg Functional Functional No Conversion <10 copies Source of Protein: A recombinant E. coli strain carrying the Klenow Fragment gene. Unit Definition: 1 unit is defined as the amount of polymerase required to convert 10 nmol of dNTPs into acid insoluble material in 30 minutes at 37°C. Molecular weight: 68,202 Daltons Quality Control Analysis: Unit Activity is measured using a 2-fold serial dilution method. Dilutions of enzyme were made in a 50% glycerol Klenow (3’-5’ exo-) storage solution and added to 50 µL reactions containing Calf Thymus DNA, 1X Klenow Reaction Buffer, 3H-dTTP and 100 µM dNTPs. Reactions were incubated 10 minutes at 37°C, plunged on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26). Protein Concentration (OD280) is determined by OD280 absorbance.
    [Show full text]
  • DNA Bound by the Oxytricha Telomere Protein Is Accessible to Telomerase and Other DNA Polymerases DOROTHY E
    Proc. Natl. Acad. Sci. USA Vol. 91, pp. 405-409, January 1994 Biochemistry DNA bound by the Oxytricha telomere protein is accessible to telomerase and other DNA polymerases DOROTHY E. SHIPPEN*, ELIZABETH H. BLACKBURNt, AND CAROLYN M. PRICE0§ tDepartment of Microbiology and Immunology, University of California, San Francisco, CA 94143; and tDepartment of Chemistry, University of Nebraska, Lincoln, NB 68588 Contributed by Elizabeth H. Blackburn, August 25, 1993 ABSTRACT Macronuclear telomeres in Oxytricha exist as oftelomere protein in these two populations is not altered by DNA-protein complexes in which the termini of the G-rich additional nuclease treatment. strands are bound by a 97-kDa telomere protein. During The fragment of DNA bound by the majority of telomere telome'ic DNA replication, the replication machinery must protein molecules corresponds to the most terminal 13 or 14 have access to the G-rich strand. However, given the stability nucleotides of the T4G4T4G4 overhang (4). Dimethyl sulfate of telomere protein binding, it has been unclear how this is footprinting demonstrated that the complex formed between accomplished. In this study we investigated the ability of the telomere protein and the residual DNA fragment retains several different DNA polymerases to access telomeric DNA in the same DNA-protein contacts present at native telomeres Oxytricha telomere protein-DNA complexes. Although DNA (4). Thus, these telomeric DNA-protein complexes are useful bound by the telomere protein is not degraded by micrococcal substrates for in vitro investigations of telomere structure nuclease or labeled by terminal deoxynucleotidyltrnsferase, (10). In this study we have employed the DNA-protein this DNA serves as an efficient primer for the addition of complexes to analyze the interaction of protein-bound telo- telomeric repeats by telomerase, a specialized RNA-dependent meric DNA with components of the DNA replication ma- DNA polymerase (ribonucleoprotein reverse tanscriptase), chinery.
    [Show full text]
  • Arthur Kornberg Discovered (The First) DNA Polymerase Four
    Arthur Kornberg discovered (the first) DNA polymerase Using an “in vitro” system for DNA polymerase activity: 1. Grow E. coli 2. Break open cells 3. Prepare soluble extract 4. Fractionate extract to resolve different proteins from each other; repeat; repeat 5. Search for DNA polymerase activity using an biochemical assay: incorporate radioactive building blocks into DNA chains Four requirements of DNA-templated (DNA-dependent) DNA polymerases • single-stranded template • deoxyribonucleotides with 5’ triphosphate (dNTPs) • magnesium ions • annealed primer with 3’ OH Synthesis ONLY occurs in the 5’-3’ direction Fig 4-1 E. coli DNA polymerase I 5’-3’ polymerase activity Primer has a 3’-OH Incoming dNTP has a 5’ triphosphate Pyrophosphate (PP) is lost when dNMP adds to the chain E. coli DNA polymerase I: 3 separable enzyme activities in 3 protein domains 5’-3’ polymerase + 3’-5’ exonuclease = Klenow fragment N C 5’-3’ exonuclease Fig 4-3 E. coli DNA polymerase I 3’-5’ exonuclease Opposite polarity compared to polymerase: polymerase activity must stop to allow 3’-5’ exonuclease activity No dNTP can be re-made in reversed 3’-5’ direction: dNMP released by hydrolysis of phosphodiester backboneFig 4-4 Proof-reading (editing) of misincorporated 3’ dNMP by the 3’-5’ exonuclease Fidelity is accuracy of template-cognate dNTP selection. It depends on the polymerase active site structure and the balance of competing polymerase and exonuclease activities. A mismatch disfavors extension and favors the exonuclease.Fig 4-5 Superimposed structure of the Klenow fragment of DNA pol I with two different DNAs “Fingers” “Thumb” “Palm” red/orange helix: 3’ in red is elongating blue/cyan helix: 3’ in blue is getting edited Fig 4-6 E.
    [Show full text]
  • Processivity of DNA Polymerases: Two Mechanisms, One Goal Zvi Kelman1*, Jerard Hurwitz1 and Mike O’Donnell2
    Minireview 121 Processivity of DNA polymerases: two mechanisms, one goal Zvi Kelman1*, Jerard Hurwitz1 and Mike O’Donnell2 Replicative DNA polymerases are highly processive Processive DNA synthesis by cellular replicases and the enzymes that polymerize thousands of nucleotides without bacteriophage T4 replicase dissociating from the DNA template. The recently Until recently, the only mechanism for high processivity determined structure of the Escherichia coli bacteriophage that was understood in detail was that utilized by cellular T7 DNA polymerase suggests a unique mechanism that replicases and the replicase of bacteriophage T4. This underlies processivity, and this mechanism may generalize mechanism involves a ring-shaped protein called a ‘DNA to other replicative polymerases. sliding clamp’ that encircles the DNA and tethers the polymerase catalytic unit to the DNA [3,4]. The three- Addresses: 1Department of Molecular Biology, Memorial Sloan- dimensional structures of several sliding clamps have been Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, 2 determined: the eukaryotic proliferating cell nuclear USA and Laboratory of DNA Replication, Howard Hughes Medical β Institute, The Rockefeller University, 1230 York Avenue, New York, NY antigen (PCNA) [5,6]; the subunit of the prokaryotic 10021, USA. DNA polymerase III [7]; and the bacteriophage T4 gene 45 protein (gp45) (J Kuriyan, personal communication) *Corresponding author. (Figure 1). The overall structure of these clamps is very E-mail: [email protected] similar; the PCNA, β subunit and gp45 rings are super- Structure 15 February 1998, 6:121–125 imposable [8]. Each ring has similar dimensions and a http://biomednet.com/elecref/0969212600600121 central cavity large enough to accommodate duplex DNA (Figure 1).
    [Show full text]
  • Reverse Transcriptase Activity Innate to DNA Polymerase I and DNA
    Reverse transcriptase activity innate to DNA SEE COMMENTARY polymerase I and DNA topoisomerase I proteins of Streptomyces telomere complex Kai Bao*† and Stanley N. Cohen*‡§ Departments of *Genetics and ‡Medicine, Stanford University School of Medicine, Stanford, CA 94305-5120 Edited by Nicholas R. Cozzarelli, University of California, Berkeley, CA, and approved August 10, 2004 (received for review June 18, 2004) Replication of Streptomyces linear chromosomes and plasmids gation and, remarkably, that both of these eubacterial enzymes -proceeds bidirectionally from a central origin, leaving recessed 5؅ can function efficiently as RTs in addition to having the bio termini that are extended by a telomere binding complex. This chemical properties predicted from their sequences. We further complex contains both a telomere-protecting terminal protein show that the Streptomyces coelicolor and Streptomyces lividans (Tpg) and a telomere-associated protein that interacts with Tpg TopA proteins are prototypes for a subfamily of bacterial and the DNA ends of linear Streptomyces replicons. By using topoisomerases whose catalytic domains contain a unique Asp– histidine-tagged telomere-associated protein (Tap) as a scaffold, Asp doublet motif that is required for their RT activity and which we identified DNA polymerase (PolA) and topoisomerase I (TopA) is essential also to the RNA-dependent DNA polymerase func- proteins as other components of the Streptomyces telomere com- tions of HIV RT and eukaryotic cell telomerases (16, 17). plex. Biochemical characterization of these proteins indicated that both PolA and TopA exhibit highly efficient reverse transcriptase Materials and Methods (RT) activity in addition to their predicted functions. Although RT Plasmid and Bacterial Strains.
    [Show full text]
  • Molecular Insights Into Mitochondrial Transcription and Its Role in DNA Replication
    Molecular insights into mitochondrial transcription and its role in DNA replication Viktor Posse Department of Medical Biochemistry and Cell Biology Institute of Biomedicine Sahlgrenska Academy University of Gothenburg Gothenburg, Sweden, 2017 Molecular insights into mitochondrial transcription and its role in DNA replication © 2016 Viktor Posse [email protected] ISBN 978-91-629-0024-3 (PRINT) ISBN 978-91-629-0023-6 (PDF) http://hdl.handle.net/2077/48657 Printed in Gothenburg, Sweden 2016 Ineko AB Abstract The mitochondrion is an organelle of the eukaryotic cell responsible for the production of most of the cellular energy-carrying molecule adenosine triphosphate (ATP), through the process of oxidative phosphorylation. The mitochondrion contains its own genome, a small circular DNA molecule (mtDNA), encoding essential subunits of the oxidative phosphorylation system. Initiation of mitochondrial transcription involves three proteins, the mitochondrial RNA polymerase, POLRMT, and its two transcription factors, TFAM and TFB2M. Even though the process of transcription has been reconstituted in vitro, a full molecular understanding is still missing. Initiation of mitochondrial DNA replication is believed to be primed by transcription prematurely terminated at a sequence known as CSBII. The mechanisms of replication initiation have however not been fully defined. In this thesis we have studied transcription and replication of mtDNA. In the first part of this thesis we demonstrate that the transcription initiation machinery is recruited in discrete steps. Furthermore, we find that a large domain of POLRMT known as the N-terminal extension is dispensable for transcription initiation, and instead functions in suppressing initiation events from non-promoter DNA. Additionally we demonstrate that TFB2M is the last factor that is recruited to the initiation complex and that it induces melting of the mitochondrial promoters.
    [Show full text]
  • High-Level Expression, Purification, and Thermus Aquatlcus DNA
    Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press High-level Expression, Purification, and Enzymatic Characterization of Full-length Thermus aquatlcus DNA Polymerase and a Truncated Form Deficient in 5' to 3' Exonuclease Activity Frances C. Lawyer, 1 Susanne Stoffel, 1 Randall K. Saiki, 2 Sheng-Yung Chang, 3 Phoebe A. Landre, ~ Richard D. Abrarnson, 1 and David H. Gelfand 1 1Program in Core Research and Departments of 2Human Genetics and 3Infectious Disease, Roche Molecular Systems, Alameda, California 94501 The Thermus aquaticus DNA poly- life of 9 min at 97.5~ The Stoffel I in the native host is quite low (0.01- merase I (Taq Pol I) gene was cloned fragment has a half-life of 21 min at 0.02% of total protein). The cloning and into a plasmid expression vector that 97.5~ Taq Pol I contains a polymer- expression of full-length 94-kD Taq Pol I utilizes the strong bacteriophage ization-dependent 5' to 3' exonu- in E. coli under control of the E. coli lac PL promoter. A truncated form of Taq clease activity whereas the Stoffel promoter r or the tac promoter (7~ has Pol I was also constructed. The two fragment, deleted for the 5' to 3' ex- been reported. Because polymerase constructs made it possible to com- onuclease domain, does not possess yields in these constructs were low pare the full-length 832-amino-acid that activity. A comparison is made (-0.01% of total protein in our initial Taq Pol I and a deletion derivative among thermostable DNA poly- construct; see ref.
    [Show full text]
  • Family a and B DNA Polymerases in Cancer: Opportunities for Therapeutic Interventions
    biology Review Family A and B DNA Polymerases in Cancer: Opportunities for Therapeutic Interventions Vinit Shanbhag 1,2, Shrikesh Sachdev 2,3, Jacqueline A. Flores 2,3, Mukund J. Modak 4 and Kamalendra Singh 2,3,4,5,* 1 Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA; [email protected] 2 The Christopher S. Bond Life Science Center, University of Missouri, Columbia, MO 65211, USA; [email protected] (S.S.); [email protected] (J.A.F.) 3 Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65211, USA 4 Department of Microbiology, Biochemistry and Molecular Genetics 225 Warren Street, NJ 07103, USA; [email protected] 5 Department of Laboratory Medicine, Karolinska Institutet, Stockholm 141 86, Sweden * Correspondence: [email protected]; Tel.: +1-573-882-9024 Received: 13 November 2017; Accepted: 29 December 2017; Published: 2 January 2018 Abstract: DNA polymerases are essential for genome replication, DNA repair and translesion DNA synthesis (TLS). Broadly, these enzymes belong to two groups: replicative and non-replicative DNA polymerases. A considerable body of data suggests that both groups of DNA polymerases are associated with cancer. Many mutations in cancer cells are either the result of error-prone DNA synthesis by non-replicative polymerases, or the inability of replicative DNA polymerases to proofread mismatched nucleotides due to mutations in 30-50 exonuclease activity. Moreover, non-replicative, TLS-capable DNA polymerases can negatively impact cancer treatment by synthesizing DNA past lesions generated from treatments such as cisplatin, oxaliplatin, etoposide, bleomycin, and radiotherapy. Hence, the inhibition of DNA polymerases in tumor cells has the potential to enhance treatment outcomes.
    [Show full text]