DOCSLIB.ORG

Interactions of Dna Polymerase Theta and Ku70/80 With

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Interactions of Dna Polymerase Theta and Ku70/80 With INTERACTIONS OF DNA POLYMERASE THETA AND KU70/80 WITH OXIDATIVE DNA DAMAGE by Daniel Laverty A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, MD Submitted June 2018 Abstract Oxidized abasic sites (L, C4-AP, and DOB) are formed by ionizing radiation, reactive oxygen species, and some chemotherapeutics. Like abasic sites (AP), these lesions are cytotoxic and mutagenic and must be repaired, primarily by base excision repair (BER). If left unrepaired, abasic lesions stall replication and induce mutations. Repair of oxidized abasic lesions exhibits unique challenges, however. C4-AP and DOB inactivate the lyase activity of the repair enzymes DNA polymerase β and λ. Recently, several other enzymes were shown to possess lyase activity, allowing them to excise abasic lesions. Among these are DNA polymerase θ (Pol θ) and Ku70/80 (Ku). As Pol θ promotes resistance to cancer therapies which form oxidized abasic sites, the repair and replication of these lesions by Pol θ is potentially important. Ku is a core factor for non-homologous end-joining and removes AP from double strand breaks (DSBs). The interaction of Ku with oxidized abasic sites near DSB termini is potentially important for the response to ionizing radiation, which is used as a cancer treatment. Synthetic oligonucleotides containing abasic and oxidized abasic sites were prepared, and their repair or replication by Pol θ was analyzed. Pol θ bypasses C4-AP and L with reduced efficiency relative to AP and has a strong propensity to induce frameshift mutations during bypass of AP, C4-AP, L, and the oxidized nucleobase, thymidine glycol. Studies on the repair of C4-AP and DOB by Pol θ showed that Pol θ is inactivated by pC4- AP, which covalently modifies Lys2383, but not by DOB. Site-directed mutagenesis showed that Lys2383 is essential for both polymerase and lyase activity of Pol θ. These results have unveiled the primary nucleophile (Lys2383) responsible for Pol θ lyase activity. ii The repair of oxidized abasic sites by Ku was also analyzed. Ku exhibits differential repair capability on different oxidized abasic lesions. C4-AP and DOB are excised more efficiently than AP, yet Ku cannot excise L. Failure to remove this lesion can potentially inhibit repair of a DSB. Unlike Pol θ which was modified by pC4-AP, modification of Ku was not detected following repair of DOB or C4-AP. Advisor: Prof. Marc M. Greenberg Readers: Prof. Steven Rokita Prof Rebecca Klausen iii Acknowledgements I’m very thankful to all the people who have helped me through this journey. My parents, my brother, and my sister have always been extremely supportive, and I couldn’t have done it without them. Thanks to all my friends here in Baltimore and my friends back home. Thanks to all the members of the Greenberg lab, past and present, for their tremendous help. Special thanks to Liwei Weng and to Rakesh for their great help when I was getting started in the lab. Thanks very much to Kun for his help with cloning and protein purification and for helpful discussions. Thanks to Arnab and Josh for their friendship, support, and advice. I’m also very grateful to my friend Liwei Zheng who’s been here every step of the way. I couldn’t have asked for a better friend to have by my side on this journey. Thanks to Shelby and Marco for their support and for a lot of laughs along the way. Thanks to Sahil for support and encouragement. Thanks to all other members of the lab, past and present, for their support. I’m very thankful to Dr. Greenberg for his mentorship through this journey. I’m continually impressed by his dedication to ensuring that his students get the most out of their graduate experience. I’m especially thankful for his encouragement in pursuing my interests when they differed from our original goals. Thanks to the readers of my thesis, Dr. Rokita and Dr. Klausen. Thanks to Dr. Townsend for encouragement and advice during my search for a post-doctoral position. iv Table of Contents Abstract .............................................................................................................................. ii Acknowledgements .......................................................................................................... iv List of Tables ..................................................................................................................... x List of Figures. ................................................................................................................. xii List of Abbreviations ..................................................................................................... xix 1. Introduction ............................................................................................................... 1 2. Background ............................................................................................................... 3 2.1. DNA Damage .......................................................................................................... 3 2.1.1 Abasic Sites ....................................................................................................... 4 2.1.2 Oxidative DNA Damage .................................................................................. 5 2.1.2.1 Thymidine Glycol .......................................................................................... 6 2.1.2.2 Sugar Oxidation ............................................................................................ 8 2.1.2.3 C4’-Oxidized Abasic Site .............................................................................. 9 2.1.2.4 2-Deoxyribonolactone ................................................................................. 11 2.2 Double Strand Breaks .......................................................................................... 13 2.2.1 Double Strand Break Formation by Ionizing Radiation ............................ 14 2.2.2 Double Strand Break Formation by Antitumor Antibiotics ...................... 15 2.2.3 Repair of Double Strand Breaks .................................................................. 17 2.2.3.1 Non-Homologous End Joining ................................................................... 17 v 2.2.3.2 Homologous Recombination ...................................................................... 21 2.2.3.3 Alternative End Joining ............................................................................. 22 2.3.1 Mechanism of Base Excision Repair ............................................................ 24 2.3.2 Substrate Scope of Base Excision Repair .................................................... 26 2.3.3 Glycosylases .................................................................................................... 27 2.3.4 Ape1 ................................................................................................................. 29 2.3.5 Base Excision Repair Polymerases ............................................................... 29 2.3.6 Base Excision Repair of Oxidized Abasic Sites ........................................... 32 2.4 DNA Damage Tolerance by Translesion Synthesis ........................................... 34 2.4.1 Mechanism of Translesion Synthesis ........................................................... 35 2.4.2 Discovery of Translesion Synthesis Polymerases ........................................ 37 2.4.3 Translesion Synthesis Polymerases in Eukaryotes ..................................... 38 2.4.3.1 General Features of Translesion Synthesis Polymerases ........................ 39 2.4.3.2 Rev1 .............................................................................................................. 41 2.4.3.3 Pol η .............................................................................................................. 42 2.4.3.4 Pol κ .............................................................................................................. 43 2.4.3.5 Pol ι ............................................................................................................... 45 2.4.3.6 Pol ζ .............................................................................................................. 46 2.4.3.7 Other Polymerases Involved in Translesion Synthesis ............................ 47 2.4.4 Translesion Synthesis of Abasic Sites and Oxidized Abasic Sites ............. 49 vi 2.4.5 Translesion Synthesis of Thymidine Glycol ................................................ 53 2.4.6 Regulation of Translesion Synthesis in Eukaryotes .................................... 54 2.5 DNA Polymerase Theta (Pol θ) ............................................................................ 56 2.5.1 Discovery and Cloning ................................................................................... 56 2.5.2 Structure and Unique Characteristics of Pol θ ........................................... 58 2.5.3 Function of Pol θ ............................................................................................ 59 2.5.4 Pol θ as a Cancer Target ............................................................................... 62 2.6 Ku70/80 .................................................................................................................. 63 2.6.1 Ku Structure ................................................................................................... 64 2.6.2 Structural Role of Ku in NHEJ ...................................................................
Load more

Recommended publications
  • Biochemical Studies of Dna Polymerase Theta A
    BIOCHEMICAL STUDIES OF DNA POLYMERASE THETA A Dissertation Submitted to the Temple University Graduate Board In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY by Ahmet Y Ozdemir May 2019 Examining Committee Members: Richard T Pomerantz,PhD, Advisory Chair, Fels Institute for Cancer Research and Molecular Biology & Medical Genetics and Molecular Biochemistry Xavier Graña-Amat,PhD, Fels Institute for Cancer Research and Molecular Biology & Medical Genetics and Molecular Biochemistry Tomasz Skorski, MD, PhD, DSc, Fels Institute for Cancer Research and Molecular Biology & Microbiology and Immunology Italo Tempera, PhD, Fels Institute for Cancer Research and Molecular Biology & Microbiology and Immunology Alexander Mazin, PhD, External Member, Biochemistry & Molecular Biology, Drexel University © Copyright 2019 by Ahmet Y Ozdemir All Rights Reserved ii ABSTRACT POLQ is a unique multifunctional replication and repair gene that encodes a multidomain protein with a N-terminal superfamily 2 helicase and a C-terminal A-family polymerase. Although the function of the polymerase domain has been investigated, little is understood regarding the helicase domain. Multiple studies have reported that polymerase θ-helicase (Polθ-helicase) is unable to unwind DNA. However, it exhibits ATPase activity that is stimulated by single-stranded DNA, which presents a biochemical conundrum. In contrast to previous reports, we demonstrate that Polθ-helicase (residues 1– 894) efficiently unwinds DNA with 3'–5' polarity, including DNA with 3' or 5' overhangs, blunt- ended DNA, and replication forks. Polθ-helicase also efficiently unwinds RNA- DNA hybrids and exhibits a preference for unwinding the lagging strand at replication forks, similar to related HELQ helicase. Finally, we find that Polθ-helicase can facilitate strand displacement synthesis by Polθ-polymerase, suggesting a plausible function for the helicase domain.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2014/0079836A1 Mcdaniel (43) Pub
    US 20140079836A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0079836A1 McDaniel (43) Pub. Date: Mar. 20, 2014 (54) METHODS AND COMPOSITIONS FOR (52) U.S. Cl. ALTERING HEALTH, WELLBEING AND CPC ............... A61K 36/74 (2013.01); A61 K3I/122 LIFESPAN (2013.01) USPC ............. 424/777; 514/690: 435/375; 506/16; (71) Applicant: LifeSpan Extension, LLC, Virginia 435/6.12 Beach, VA (US) (72) Inventor: David H. McDaniel, Virginia Beach, VA (57) ABSTRACT (US) Described herein are the results of comprehensive genetic (73) Assignee: LifeSpan Extension, LLC expression and other molecular analysis p the effect s anti oxidants on biological systems, including specifically differ (21) Appl. No.: 14/084,553 ent human cells. Based on these analyses, methods and com (22) Filed: Nov. 19, 2013 positions are described for modifying or influencing the lifespan of cells, tissues, organs, and organisms. In various Related U.S. Application Data embodiments, there are provided methods for modulating the activity of the gene maintenance process in order to influence (60) Continuation of application No. 13/898.307, filed on the length and/or structural integrity of the telomere in living May 20, 2013, which is a division of application No. cells, as well as methods for modulating the rate/efficiency of 12/629,040, filed on Dec. 1, 2009, now abandoned. the cellular respiration provided by the mitochondria, mito (60) Provisional application No. 61/118,945, filed on Dec. chondrial biogenesis, and maintenance of the mitochondrial 1, 2008. membrane potential. Exemplary lifespan altering compounds include natural and synthetic antioxidants, such as plant anti Publication Classification oxidant and polyphenol compounds derived from coffee cherry, tea, berry, and so forth, including but not limited to (51) Int.
    [Show full text]
  • The Central Role of Acetyl-Coa in Plant Metabolism, As Examined Through Studies of ATP Citrate Lyase and the Bio1 Mutant of Arabidopsis Elizabeth K
    Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2004 The central role of acetyl-CoA in plant metabolism, as examined through studies of ATP citrate lyase and the bio1 mutant of Arabidopsis Elizabeth K. Winters Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Genetics Commons, Molecular Biology Commons, and the Plant Sciences Commons Recommended Citation Winters, Elizabeth K., "The ec ntral role of acetyl-CoA in plant metabolism, as examined through studies of ATP citrate lyase and the bio1 mutant of Arabidopsis " (2004). Retrospective Theses and Dissertations. 1203. https://lib.dr.iastate.edu/rtd/1203 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. The central role of acetyl-CoA in plant metabolism, as examined through studies of ATP citrate lyase and the biol mutant of Arabidopsis by Elizabeth K. Winters A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Plant Physiology Program of Study Committee: Eve Syrkin Wurtele, Co-major Professor Basil J. Nikolau, Co-major Professor Thomas Baum James T. Colbert David J. Oliver Mark Westgate Iowa State University Ames, Iowa 2004 UMI Number: 3158378 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted.
    [Show full text]
  • The Dark Side of UV-Induced DNA Lesion Repair
    G C A T T A C G G C A T genes Review The Dark Side of UV-Induced DNA Lesion Repair Wojciech Strzałka 1, Piotr Zgłobicki 1, Ewa Kowalska 1, Aneta Ba˙zant 1, Dariusz Dziga 2 and Agnieszka Katarzyna Bana´s 1,* 1 Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland; [email protected] (W.S.); [email protected] (P.Z.); [email protected] (E.K.); [email protected] (A.B.) 2 Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-12-664-6410 Received: 27 October 2020; Accepted: 29 November 2020; Published: 2 December 2020 Abstract: In their life cycle, plants are exposed to various unfavorable environmental factors including ultraviolet (UV) radiation emitted by the Sun. UV-A and UV-B, which are partially absorbed by the ozone layer, reach the surface of the Earth causing harmful effects among the others on plant genetic material. The energy of UV light is sufficient to induce mutations in DNA. Some examples of DNA damage induced by UV are pyrimidine dimers, oxidized nucleotides as well as single and double-strand breaks. When exposed to light, plants can repair major UV-induced DNA lesions, i.e., pyrimidine dimers using photoreactivation. However, this highly efficient light-dependent DNA repair system is ineffective in dim light or at night. Moreover, it is helpless when it comes to the repair of DNA lesions other than pyrimidine dimers.
    [Show full text]
  • Wo 2010/065567 A2
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 10 June 2010 (10.06.2010) WO 2010/065567 A2 (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every A61K 36/889 (2006.01) A61K 31/16 (2006.01) kind of national protection available): AE, AG, AL, AM, A61K 36/736 (2006.01) A61K 31/05 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, A61K 31/166 (2006.01) A61P 5/00 (2006.01) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, PCT/US2009/066294 KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (22) International Filing Date: ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 1 December 2009 (01 .12.2009) NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, (25) Filing Language: English TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 61/1 18,945 1 December 2008 (01 .12.2008) US GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (71) Applicant (for all designated States except US): LIFES¬ TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, PAN EXTENSION LLC [US/US]; 933 First Colonial ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, Road, Suite 114, Virginia Beach, VA 23454 (US).
    [Show full text]
  • Investigation of Dna Damage Response at Interstitial Telomeric Sites in Chinese Hamster Cell Lines
    INVESTIGATION OF DNA DAMAGE RESPONSE AT INTERSTITIAL TELOMERIC SITES IN CHINESE HAMSTER CELL LINES A thesis submitted for a degree of Doctor of Philosophy by Sheila Matta Castillo College of Health and Life Sciences Department of Life Sciences Brunel University July 2018 Declaration I hereby declare that the research presented in this thesis is my own work, except where otherwise specified, and has not been submitted for any other degree. Sheila Matta July 2018 2 Abstract Telomeres are highly specialised structures that protect the ends of linear chromosomes from damage. Telomeric DNA consists of tandem repeats of a specific sequence (TTAGGG)n that varies in length depending on the species. Two telomere maintenance mechanisms are known to exist. Telomerase, an enzyme that synthesises new tandem repeats onto the end of linear chromosome and ALT (Alternative Lengthening of Chromosomes), which uses recombination to lengthen telomeric sequences. In some mammalian species, both mechanisms are known to co-exist. Telomeric DNA that is found at non-telomere sites are referred to as interstitial telomeric sequences (ITS). Several studies have shown that ITS sites in the Chinese hamster are preferred sites for radiation and spontaneous chromosomal damage. Once damage occurs, repair mechanisms such as HR (homologous recombination) and NHEJ (non-homologous end joined) are activated. However, the contribution to this repair by telomerase and ALT is unknown. The aim of this work therefore is to investigate the contribution that above mention mechanisms (HR, NHEJ, telomerase and ALT) make to ITS repair in Chinese hamster cells. Classical cytogenetic techniques such as Giemsa staining, chromosomal aberration analysis, FISH (Fluorescence in situ hybridazation, IHC (Immunohistochemistry), TIF (Telomere dysfunction induced foci), were used to quantify chromosomal damage after IR (ionising radiation) treatment of normal and repair deficient (HR and NHEJ) Chinese 3 hamster cell lines.
    [Show full text]
  • Small‐Molecule Inhibitors of Dna Polymerase Function
    SMALL‐MOLECULE INHIBITORS OF DNA POLYMERASE FUNCTION DISSERTATION zur Erlangung des Akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Tobias Strittmatter aus Küssaberg‐Kadelburg an der Universität Konstanz Mathematisch‐Naturwissenschaftliche Sektion Fachbereich Chemie 2014 Tag der mündlichen Prüfung: 05. Dezember 2014 Prüfungsvorsitz: Prof. Dr. Gerhard Müller 1. Referent: Prof. Dr. Andreas Marx 2. Referent: Prof. Dr. Thomas U. Mayer Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-274434 „Meinen Eltern” Danksagung Die vorliegende Arbeit entstand in der Zeit von November 2009 bis August 2014 in der Arbeitsgruppe von Prof. Dr. Andreas Marx am Lehrstuhl für Organische und Zelluläre Chemie am Fachbereich Chemie der Universität Konstanz. Nach diesem Zeitraum geprägt von intensiver Forschungsarbeit liegt nun meine Dissertation vor und ein weiteres Kapitel meiner beruflichen Karriere kann nun erfolgreich abgeschlossen werden. Aus diesem Grund ist es jetzt auch an der Zeit, mich bei all denen zu bedanken, die mich während dieser spannenden Zeit begleitet, unterstützt und gefördert haben. Meinem Doktorvater Prof. Dr. Andreas Marx danke ich ganz herzlich für die Überlassung der sehr interessanten und interdisziplinären Themenstellung, sowie für das in mich gesetzte Vertrauen, welches mir viel Raum für die selbstständige Bearbeitung und kreative Gestaltung des Themas erlaubte. Ihm, wie auch den weiteren Mitgliedern meines „Thesis Committee“, Prof. Dr. Thomas Mayer und Herrn Prof. Dr. Michael Berthold, danke ich zudem für die anregenden wissenschaftlichen Diskussionen und die geleisteten Hilfestellungen. Prof. Dr. Thomas Mayer danke ich außerdem für die Übernahme des Zweitgutachtens und Herrn Prof. Dr. Gerhard Müller für die Übernahme des Prüfungsvorsitzes.
    [Show full text]
  • Phosphorylation Regulates Human Pol Stability and Damage Bypass Throughout the Cell Cycle
    Published online 24 July 2017 Nucleic Acids Research, 2017, Vol. 45, No. 16 9441–9454 doi: 10.1093/nar/gkx619 Phosphorylation regulates human pol␩ stability and damage bypass throughout the cell cycle Federica Bertoletti1,†, Valentina Cea1,†, Chih-Chao Liang2, Taiba Lanati1, Antonio Maffia1,3, Mario D.M. Avarello1, Lina Cipolla1, Alan R. Lehmann4, Martin A. Cohn2 and Simone Sabbioneda1,* 1Istituto di Genetica Molecolare-CNR, 27100, Pavia, Italy, 2Department of Biochemistry, University of Oxford, OX1 3QU, Oxford, UK, 3Dipartimento di Biologia e Biotecnologie ‘Lazzaro Spallanzani’, Universita` degli Studi di Pavia, 27100, Pavia, Italy and 4Genome Damage and Stability Centre, University of Sussex, BN1 9RQ, Brighton, UK Received April 26, 2017; Revised July 05, 2017; Editorial Decision July 06, 2017; Accepted July 06, 2017 ABSTRACT a distorted template (1). The majority of TLS polymerases belong to the Y family and include pol␩, pol␫, pol␬ and DNA translesion synthesis (TLS) is a crucial dam- Rev1 along with the B-family member pol␨ (1). The Y fam- age tolerance pathway that oversees the completion ily polymerases can bypass the damage by virtue of a more of DNA replication in the presence of DNA damage. open catalytic site that can accommodate DNA lesions, TLS polymerases are capable of bypassing a dis- such as cyclobutane pyrimidine dimers (CPDs), created by torted template but they are generally considered in- exposure to UV light (2). As a trade-off, this wider catalytic accurate and they need to be tightly regulated. We site is prone to errors during nucleotide incorporation, so have previously shown that pol␩ is phosphorylated TLS polymerases need to be tightly controlled.
    [Show full text]
  • CATALOG of ANTIBODIES for 293T Cells Using NB100-214
    Western blot analysis of CATALOG OF ANTIBODIES FOR 293T cells using NB100-214. Species: Hu Applications: IP, WB DNA REPAIR Immuno- fluorescent staining of SiHa cells using NB100-182. Species: Hu, Mu Applications: IF, IHC-P, IP, WB Western blot analysis of FANCG transfected COS1 cell lysate using NB100-2566. Species: Hu Applications: WB Western blot analysis of: (1) MCF-7 lysate (2) HeLa lysate (3) 293 lysate and (4) SKOV3 lysate using NB100-416. Species: Hu Applications: WB, IP Western blot analysis of 293T whole cell lysate using NB100-472. Species: Hu Applications: IP, WB Table of Contents DNA Repair Non-Homologous End Joining ...2 DNA repair refers to a collection of processes by which a cell identifies and Homologous Recombination .. 3-4 corrects damage to the DNA molecules that encode its genome. In human Checkpoint Signaling & Double Strand Break Repair .. 5-7 cells, both normal metabolic activities and environmental factors (such as UV Base Excision Repair ..................8 light) can cause DNA damage, resulting in up to half a million individual Nucleotide Excision ...................9 molecular lesions per cell per day. Many of these lesions cause structural Mismatch Repair .................... 10 damage to the DNA molecule and can alter or eliminate the cell’s ability DNA Polymerase .................... 11 to transcribe the gene that the affected DNA encodes. Other lesions induce Direct Reversal of Damage ..... 11 potentially harmful mutations in the cell’s genome, which affect the survival Longevity ............................... 12 of its daughter cells after it undergoes mitosis. Consequently, the DNA repair Syndromes Linked to process must be constantly active so that it can respond quickly to any damage DNA Repair Defects ..............
    [Show full text]
  • Open Mohammad Almishwat MS 2013
    The Pennsylvania State University The Graduate School Eberly College of Science TIME-RESOLVED X-RAY CRYSTALLOGRAPHY OF DNA POLYMERASES: A PLATFORM FOR STUDIES IN STRUCTURAL AND MECHANISTIC ENZYMOLOGY A Thesis in Biochemistry, Microbiology, and Molecular Biology by Mohammad A. Almishwat © 2013 Mohammad A. Almishwat Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2013 The thesis of Mohammad A. Almishwat was reviewed and approved* by the following: Katsuhiko Murakami Associate Professor of Biochemistry and Molecular Biology Thesis Co-Adviser J. Gregory Ferry Stanley Person Professor of Biochemistry and Molecular Biology Thesis Co-Adviser Susan Hafenstein Assistant Professor of Medicine, Microbiology and Immunology Paul Babitzke Professor of Biochemistry and Molecular Biology Co-Director of Graduate Studies *Signatures are on file in the Graduate School. ii Abstract DNA-dependent DNA polymerase is the enzyme responsible for carrying out DNA replication. DNA polymerase regulates the faithful transmission of an organism’s genetic material, and performs various tasks involved in correcting errors that occur during the process of DNA replication. Extensive genetic and biochemical research has been done to characterize the general mechanism of DNA replication and the factors that govern its fidelity, and yet the mechanistic details of this fidelity, how a correct deoxynucleotide is selected for by the enzyme for incorporation, and an incorrect one is excluded, is yet to be determined. Structural studies of various DNA polymerases and their complexes with DNA have provided a great deal of insight into how catalysis in nucleotide incorporation occurs, and has also provided empirical models of how fidelity is brought about in these enzymes during DNA replication.
    [Show full text]
  • Ep 2336315 A2
    (19) TZZ ¥¥¥_ T (11) EP 2 336 315 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 22.06.2011 Bulletin 2011/25 C12N 15/10 (2006.01) C12Q 1/68 (2006.01) C40B 40/06 (2006.01) C40B 50/06 (2006.01) (21) Application number: 10192717.6 (22) Date of filing: 01.12.2006 (84) Designated Contracting States: • Husemoen, Birgitte Nystrup AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 2500 Valby (DK) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Dolberg, Johannes SK TR 1674 Copenhagen V (DK) Designated Extension States: • Jensen, Kim Birkebæk AL BA HR MK RS 2610 Rødovre (DK) • Petersen, Lene (30) Priority: 01.12.2005 DK 200501704 2100 Copenhagen Ø (DK) 02.12.2005 US 741490 P • Nørregaard-Madsen, Mads 3460 Birkerød (DK) (62) Document number(s) of the earlier application(s) in • Godskesen, Michael Anders accordance with Art. 76 EPC: 2950 Vedbæk (DK) 06818144.5 / 1 957 644 • Glad, Sanne Schrøder 2750 Ballerup (DK) (71) Applicant: Nuevolution A/S • Neve, Søren 2100 Copenhagen (DK) 2800 Lyngby (DK) • Thisted, Thomas (72) Inventors: 3600 Frederikssund (DK) • Franch, Thomas • Kronberg, Tine Titilola Akinleminu 3070 Snekkersten (DK) 3500 Værløse (DK) • Lundorf, Mikkel Dybro • Sams, Christian 4000 Roskilde (DK) 3500 Værløse (DK) • Jakobsen, Soren Nyboe • Felding, Jakob 2000 Frederiksberg (DK) 2920 Charlottenlund (DK) • Olsen, Eva Kampmann • Freskgård, Per-Ola 2730 Herlev (DK) 603 79 Norrkörping (SE) • Andersen, Anne Lee • Gouliaev, Alex Haahr 4100 Ringsted (DK) 3670 Veksø Sjælland (DK) • Holtmann, Anette • Pedersen, Henrik 2750 Ballerup (DK) 3540 Lynge (DK) • Hansen, Anders Holm 2400 Copenhagen NV (DK) (74) Representative: Aamand, Jesper L.
    [Show full text]
  • Insight Into the Fidelity of Two X-Family Polymerases: Dna Polymerase Mu and Dna Polymerase Beta
    INSIGHT INTO THE FIDELITY OF TWO X-FAMILY POLYMERASES: DNA POLYMERASE MU AND DNA POLYMERASE BETA DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Michelle P. Roettger, B.S. * * * * * The Ohio State University 2008 Dissertation Committee: Approved by Professor Ming-Daw Tsai, Advisor Professor Ross Dalbey, Co-advisor _________________________________ Professor Richard Swenson Advisor Ohio State Biochemistry Program Professor Juan Alfonzo ABSTRACT DNA polymerase µ (Pol µ) is a recently discovered X-family DNA polymerase, with yet unknown physiological function. Due to its preferential expression in secondary lymphoid tissues, this enzyme has been implicated as a potential mutase involved in the somatic hypermutation (SHM) of immunoglobulin (Ig) genes during antibody affinity maturation. To evaluate the hypothesis which regards Pol µ as a mutase in Ig maturation, pre-steady-state kinetic methods were used to accurately measure the fidelity of human Pol µ based on all 16 possible deoxynucleotide (dNTP) incorporations and four matched ribonucleotide (rNTP) incorporations into normal DNA primer/template substrates. The overall fidelity of Pol µ was estimated to be in the range of 10-3-10-5 for both dNTP and rNTP incorporations and was sequence-independent. Furthermore, to evaluate the template-independent polymerization of this enzyme, the kinetics of dNTP and rNTP incorporation into a single-stranded DNA substrate were measured and qualitatively compared to terminal deoxynucleotidyl transferase (TdT). The potential biological functions of Pol µ are discussed on the basis of the pre-steady-state kinetic data. DNA Polymerase β (Pol β), another X-family polymerase has been well characterized both biochemically and structurally.
    [Show full text]