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