DNA Replication

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DNA Replication Basic Reaction -- Components Needed Free Nucleotide Base (Needed for basepairing). 3’ End on the Backbone (with an Oxygen). Nucleoside TriPhosphate (NTP). Must be correct nucleotide base for basepairing. The reaction frees two of the phosphates from the nucleotide. DNA Polymerase to catalyze the 3’-5’ Phosphodiester bond. DNA Replication Polymerase w DNA Dependent DNA Polymerase w DNA Dependent RNA Polymerase w RNA Dependent DNA Polymerase w RNA Dependent RNA Polymerase DNA Dependent DNA Polymerase (DNA Polymerase) Requirements: 1. 3’ end with Oxygen on backbone 2. Unimpeded Nucleotide base on the Template Strand 3. Correct NTP (based on 2) 4. Energy requirement is met by NTP ® NMP + DP Direction: Replication goes in the 5’ to 3’ direction -- called 3’ Elongation. Proofreading DNA Polymerases can have exonuclease activity. Some can backup and redo incorrect base matches. Some are specialized to look for errors. DNA Replication Details for E. coli 1. Unwinding the DNA double helix -- Unwinding Proteins (DNA helicase); Helix Destabilizing Proteins (HDP); DNA Gyrase (DNA topoisomerase). 2. Replication Bubble. Replication takes place in replication forks on either side of the Bubble. Some replication is unidirectional (such as with Conjugation). DNA Replication Details for E. coli (cont.) 3. Replication Fork 1. DNA Polymerase I, II, III DNA Polymerase I -- Error Correcting DNA Polymerase II -- Unknown Function DNA Polymerase III -- Main Replication Enzyme 2. Synthesis of the new chromosome is bidirectional. a. Leading Strand -- 5’ to 3’ b. Lagging Strand -- 5’ to 3’ in sections (Okazaki Fragments). i) RNA Primase initiates the primer. ii) DNA Polymerase III attaches to primers and synthesizes DNA. iii) Synthesis continues through the RNA primers of the next fragment (DNA Polymerase I). iv) DNA ligase attaches the backbones of the separate fragments. DNA Replication Relationship Between Leading & Lagging Strands Polymerases form a dimer. Lagging strand loops around polymerase core units Leading and Lagging Strands are replicated together (different polymerases, but attached to each other)..

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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.
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Introduction of Human Telomerase Reverse Transcriptase to Normal Human Fibroblasts Enhances DNA Repair Capacity
Vol. 10, 2551–2560, April 1, 2004 Clinical Cancer Research 2551 Introduction of Human Telomerase Reverse Transcriptase to Normal Human Fibroblasts Enhances DNA Repair Capacity Ki-Hyuk Shin,1 Mo K. Kang,1 Erica Dicterow,1 INTRODUCTION Ayako Kameta,1 Marcel A. Baluda,1 and Telomerase, which consists of the catalytic protein subunit, No-Hee Park1,2 human telomerase reverse transcriptase (hTERT), the RNA component of telomerase (hTR), and several associated pro- 1School of Dentistry and 2Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California teins, has been primarily associated with maintaining the integ- rity of cellular DNA telomeres in normal cells (1, 2). Telomer- ase activity is correlated with the expression of hTERT, but not ABSTRACT with that of hTR (3, 4). Purpose: From numerous reports on proteins involved The involvement of DNA repair proteins in telomere main- in DNA repair and telomere maintenance that physically tenance has been well documented (5–8). In eukaryotic cells, associate with human telomerase reverse transcriptase nonhomologous end-joining requires a DNA ligase and the (hTERT), we inferred that hTERT/telomerase might play a DNA-activated protein kinase, which is recruited to the DNA role in DNA repair. We investigated this possibility in nor- ends by the DNA-binding protein Ku. Ku binds to hTERT mal human oral fibroblasts (NHOF) with and without ec- without the need for telomeric DNA or hTR (9), binds the topic expression of hTERT/telomerase. telomere repeat-binding proteins TRF1 (10) and TRF2 (11), and Experimental Design: To study the effect of hTERT/ is thought to regulate the access of telomerase to telomere DNA telomerase on DNA repair, we examined the mutation fre- ends (12, 13).
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DNA REPLICATION Objectives: 1
Lecture 1 DNA REPLICATION Objectives: 1. To understand the replication process and where it occurs in the cell cycle. 2. To describe the Replication Fork, Okazaki fragments and the enzymes involved in the unwinding process and replication. 3. To understand how DNA polymerase initiate the synthesis of new strands. 4. To define Telomeres and Telomerase and their clinical significance. Gene expression also called protein expression or often simply expression: is the process by which a gene's DNA sequence is converted into the structures and functions of a cell. The amount of protein that a cell expresses depends on: 1. the tissue, 2. the developmental stage of the organism 3. and the metabolic or physiologic state of the cell. DNA replication or DNA synthesis is the process of copying a double-stranded DNA strand, prior to cell division. The two resulting double strands are identical (if the replication went well), and each of them consists of one original and one newly synthesized strand. This is called semi conservative replication. 1 Prof. Dr. H.D.El-Yassin 2013 Lecture 1 The process of replication consists of three steps, initiation, replication and termination. 1. Prokaryotic replication Basic Requirement for DNA Synthesis 1. Substrates: the four deoxy nucleosides triphosphates are needed as substrates for DNA synthesis. Cleavage of the high-energy phosphate bond between the α and β phosphates provides the energy for the addition of the nucleotide. 2. Template: DNA replication cannot occur without a template. A template is required to direct the addition of the appropriate complementary deoxynucleotide to the newly synthesized DNA strand.
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DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli
DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Kath, James Evon. 2016. DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:26718716 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA ! ! ! ! ! ! ! DNA!polymerase!exchange!and!lesion!bypass!in!Escherichia)coli! ! A!dissertation!presented! by! James!Evon!Kath! to! The!Committee!on!Higher!Degrees!in!Biophysics! ! in!partial!fulfillment!of!the!requirements! for!the!degree!of! Doctor!of!Philosophy! in!the!subject!of! Biophysics! ! Harvard!University! Cambridge,!Massachusetts! October!2015! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ©!2015!L!James!E.!Kath.!Some!Rights!Reserved.! ! This!work!is!licensed!under!the!Creative!Commons!Attribution!3.0!United!States!License.!To! view!a!copy!of!this!license,!visit:!http://creativecommons.org/licenses/By/3.0/us! ! ! Dissertation!Advisor:!Professor!Joseph!J.!Loparo! ! ! !!!!!!!!James!Evon!Kath! ! DNA$polymerase$exchange$and$lesion$bypass$in$Escherichia)coli$ $ Abstract$ ! Translesion! synthesis! (TLS)! alleviates!
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T-Loops and the Origin of Telomeres E
PERSPECTIVES 66. Steinman, R. M., Mellman, I. S., Muller, W. A. & Cohn, Z. A. Acknowledgments eukaryotes evolved. The presence of t-loops at Endocytosis and the recycling of plasma membrane. H.S. is supported by the Norwegian Cancer Society and the J. Cell Biol. 96, 1–27 (1983). Research Council of Norway, and J.G. by the Swiss National present-day telomeres and their association 67. Lafont, F., Lecat, S., Verkade, P. & Simons, K. Annexin Science Foundation and the Human Frontier Science Programme with proteins that have evolved from RDR XIIIb associates with lipid microdomains to function in Organization. apical delivery. J. Cell Biol. 142, 1413–1427 (1998). factors might be remnants of the original 68. Aniento, F., Gu, F., Parton, R. & Gruenberg, J. Competing interests statement telomere system. Furthermore, the relative An endosomal βcop is involved in the pH-dependent The authors declare that they have no competing financial interests. formation of transport vesicles destined for late ease with which many eukaryotes can main- endosomes. J. Cell Biol. 133, 29–41 (1996). tain telomeres without telomerase might 69. Gu, F., Aniento, F., Parton, R. & Gruenberg, J. Functional Online links dissection of COP-I subunits in the biogenesis of reflect this ancient system of chromosome-end multivesicular endosomes. J. Cell Biol. 139, 1183–1195 DATABASES replication. This proposal ends with a dis- (1997). The following terms in this article are linked online to: 70. Gu, F. & Gruenberg, J. ARF1 regulates pH-dependent Interpro: http://www.ebi.ac.uk/interpro/ cussion of the advantages of the telom- COP functions in the early endocytic pathway.
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A Mutation in DNA Polymerase Α Rescues WEE1KO Sensitivity to HU
International Journal of Molecular Sciences Article A Mutation in DNA Polymerase α Rescues WEE1KO Sensitivity to HU Thomas Eekhout 1,2 , José Antonio Pedroza-Garcia 1,2 , Pooneh Kalhorzadeh 1,2, Geert De Jaeger 1,2 and Lieven De Veylder 1,2,* 1 Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; [email protected] (T.E.); [email protected] (J.A.P.-G.); [email protected] (P.K.); [email protected] (G.D.J.) 2 Center for Plant Systems Biology, VIB, 9052 Gent, Belgium * Correspondence: [email protected] Abstract: During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication and are hypersensitive to replication stress. Here, we report the identiﬁcation of the pola-2 mutant, mutated in the catalytic subunit of DNA polymerase α, as a suppressor mutant of wee1. The mutated protein appears to be less stable, causing a loss of interaction with its subunits and resulting in a prolonged S-phase. Keywords: replication stress; DNA damage; cell cycle checkpoint Citation: Eekhout, T.; Pedroza- 1. Introduction Garcia, J.A.; Kalhorzadeh, P.; De Jaeger, G.; De Veylder, L. A Mutation DNA replication is a highly complex process that ensures the chromosomes are in DNA Polymerase α Rescues correctly replicated to be passed onto the daughter cells during mitosis. Replication starts WEE1KO Sensitivity to HU. Int.
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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.
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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.
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Telomeres.Pdf
Telomeres Secondary article Elizabeth H Blackburn, University of California, San Francisco, California, USA Article Contents . Introduction Telomeres are specialized DNA–protein structures that occur at the ends of eukaryotic . The Replication Paradox chromosomes. A special ribonucleoprotein enzyme called telomerase is required for the . Structure of Telomeres synthesis and maintenance of telomeric DNA. Synthesis of Telomeric DNA by Telomerase . Functions of Telomeres Introduction . Telomere Homeostasis . Alternatives to Telomerase-generated Telomeric DNA Telomeres are the specialized chromosomal DNA–protein . Evolution of Telomeres and Telomerase structures that comprise the terminal regions of eukaryotic chromosomes. As discovered through studies of maize and somes. One critical part of this protective function is to fruitﬂy chromosomes in the 1930s, they are required to provide a means by which the linear chromosomal DNA protect and stabilize the genetic material carried by can be replicated completely, without the loss of terminal eukaryotic chromosomes. Telomeres are dynamic struc- DNA nucleotides from the 5’ end of each strand of this tures, with their terminal DNA being constantly built up DNA. This is necessary to prevent progressive loss of and degraded as dividing cells replicate their chromo- terminal DNA sequences in successive cycles of chromo- somes. One strand of the telomeric DNA is synthesized by somal replication. a specialized ribonucleoprotein reverse transcriptase called telomerase. Telomerase is required for both
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DNA Replication
9 DNA Replication To understand the chemistry Living cells must be able to duplicate their entire set of genetic instructions Goal of DNA synthesis and how every time they divide. Likewise, multicellular organisms must be able to DNA is replicated with high pass on complete copies of their genetic information to future generations. accuracy. This requires that the instructions are stored in a form that is capable of Objectives being duplicated. As we have seen (and will return to in Chapter 11), genetic instructions are embedded in the order of nucleobases in the DNA. As we After this chapter, you should be able to have also seen, the self-complementary nature of the double helix provides • describe the experiment that proved a simple (in principle) templating mechanism for replicating DNA into two that DNA replication is semi- identical copies. Only DNA (and in some instances RNA when, as in the conservative. case of the genomes of RNA viruses, it is used as a repository of genetic • diagram the reaction for information) are self-complementary and hence capable of being replicated. phosphodiester bond formation. The other three categories of macromolecules—carbohydrates, lipids, and • explain the energetics of DNA proteins—are not self-complementary and do not serve as templates for synthesis. their own production. Instead, the synthesis of these macromolecules is • explain why the 5’-to-3’ rule creates a ultimately directed by information stored in DNA through the action of conundrum during replication. enzymes and other proteins. Here we focus on the chemical and enzymatic • explain how DNA is replicated mechanisms by which DNA acts as a template for its own duplication and accurately.
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Human Glucokinase Gene
Proc. Nati. Acad. Sci. USA Vol. 89, pp. 7698-7702, August 1992 Genetics Human glucokinase gene: Isolation, characterization, and identification of two missense mutations linked to early-onset non-insulin-dependent (type 2) diabetes mellitus (glucose/metabolism/phosphorylation/structure4unctlon/chromosome 7) M. STOFFEL*, PH. FROGUELt, J. TAKEDA*, H. ZOUALItt, N. VIONNET*, S. NISHI*§, I. T. WEBER¶, R. W. HARRISON¶, S. J. PILKISII, S. LESAGEtt, M. VAXILLAIREtt, G. VELHOtt, F. SUNtt, F. lIRSt, PH. PASSAt, D. COHENt, AND G. I. BELL*"** *Howard Hughes Medical Institute, and Departments of Biochemistry and Molecular Biology, and of Medicine, The University of Chicago, 5841 South Maryland Avenue, MC1028, Chicago, IL 60637; §Second Division of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-32, Japan; IDepartment of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107; IlDepartment of Physiology and Biophysics, State University of New York, Stony Brook, NY 11794; tCentre d'Etude du Polymorphisme Humain, 27 rue Juliette Dodu, and Service d'Endocrinologie, H6pital Saint-Louis, 75010 Paris, France; and tG6ndthon, 1 rue de l'Internationale, 91000 Evry, France Communicated by Jean Dausset, May 28, 1992 ABSTRACT DNA polymorphisms in the glucokinase gene by maintaining a gradient for glucose transport into these cells have recently been shown to be tightly linked to early-onset thereby regulating hepatic glucose disposal. In (3 cells, glu- non-insulin-dependent diabetes mellitus in "80% of French cokinase is believed to be part of the glucose-sensing mech- families with this form of diabetes. We previously identified a anism and to be involved in the regulation ofinsulin secretion.
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Getting Ready for DNA Duplication
INSIGHT INTRACELLULAR ORGANIZATION Getting ready for DNA duplication The discovery of a biomolecular condensate involved in DNA replication has wide-ranging implications. NINA Y YAO AND MICHAEL E O’DONNELL involves regions with different physical proper- Related research article Parker MW, Bell ties separating from each other). The molecules M, Mir M, Kao JA, Darzacq X, Botchan MR, required for the formation of condensates are Berger JM. 2019. A new class of disordered called scaffolding factors, while the molecules elements controls DNA replication through encapsulated inside are known as clients. A initiator self-assembly. eLife 8:e48562. DOI: given condensate typically contains only those 10.7554/eLife.48562 clients involved in the relevant reaction. Now, in eLife, Michael Botchan (UC Berkeley), James Berger (Johns Hopkins) and colleagues – including Matthew Parker as first author – report on the discovery of a biomolecular condensate esearch into biomolecular condensates – required for the initiation of DNA replication in self-contained regions within cells where the fruit fly Drosophila melanogaster R specific reactions take place – is taking (Parker et al., 2019). Three proteins – ORC, cell biology by storm. Biomolecular condensates Cdc6 and Cdt1 – form the scaffold along with do not have membranes, but they are able to DNA, while a hexamer ring protein called keep the proteins and/or nucleic acids involved Mcm2-7 is the client. During the G1 phase of the in a particular reaction separate from the rest of cell cycle, the scaffold proteins load two Mcm2- the cell. Biomolecular condensates do not have 7 hexamers onto the DNA to form the pre-repli- membranes, but they are able to keep the mole- cative complex, which marks the spot where cules involved in a particular reaction (usually DNA replication will begin (Figure 1A–C; proteins, but sometimes also nucleic acids) sepa- Bell and Labib, 2016; Bleichert et al., 2017).
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