DOCSLIB.ORG

The Impact of the Integrated Stress Response on DNA Replication

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Impact of the Integrated Stress Response on DNA Replication The impact of the integrated stress response on DNA replication ____________________________________________________ Dissertation for the award of the degree "Doctor of Philosophy" (Ph.D.) Division of Mathematics and Natural Sciences of the Georg-August-Universität Göttingen within the doctoral program Molecular Biology of Cells of the Georg-August University School of Science (GAUSS) submitted by Josephine Ann Mun Yee Choo from Selangor, Malaysia Göttingen 2019 Thesis Committee 1. Prof. Dr. Matthias Dobbelstein, Institute of Molecular Oncology, University Medical Center Göttingen (UMG) 2. PD Dr. Halyna Shcherbata, Research Group – Gene Expression and Signaling, Max Planck Institute for Biophysical Chemistry (MPI-BPC) 3. Prof. Dr. Steven Johnsen, Clinic for General, Visceral and Pediatric Surgery, University Medical Center Göttingen (UMG) Members of the Examination Board 1st reviewer: Prof. Dr. Matthias Dobbelstein, Institute of Molecular Oncology, University Medical Center Göttingen (UMG) 2nd reviewer: PD Dr. Halyna Shcherbata, Research Group – Gene Expression and Signalling, Max Planck Institute for Biophysical Chemistry (MPI-BPC) External members of the Examination Board 1. Dr. Roland Dosch, Department of Developmental Biochemistry, University Medical Center Göttingen (UMG) 2. Prof. Dr. Heidi Hahn, Department of Human Genetics, University Medical Center Göttingen (UMG) 3. Prof. Dr. Dieter Kube, Department of Hematology and Oncology, University Medical Center Göttingen (UMG) 4. Dr. Nuno Raimundo, Department of Cellular Biochemistry, University Medical Center Göttingen (UMG) Date of oral examination: 12th December 2019 This thesis is dedicated to my parents. TABLE OF CONTENTS Table of Contents 1 Abstract .......................................................................................................................................... 1 2 Introduction .................................................................................................................................... 2 2.1 Central dogma of Molecular Biology ........................................................................................... 2 2.2 Translational initiation – the rate limiting step of protein synthesis ............................................. 3 2.2.1 Cap-dependent initiation ..................................................................................................... 3 2.2.2 Cap-independent initiation ................................................................................................... 5 2.2.3 Signalling pathways affecting translation initiation .............................................................. 7 2.3 The integrated stress response .................................................................................................. 8 2.3.1 ISR sensors/regulators ........................................................................................................ 8 2.3.2 eIF2alpha phosphorylation inhibits cap-dependent protein synthesis ............................... 10 2.3.3 The ISR is active. What happens then? ............................................................................ 11 2.3.4 Termination of the ISR ...................................................................................................... 12 2.3.5 The ISR and cancer .......................................................................................................... 13 2.4 DNA replication......................................................................................................................... 13 2.4.1 Regulation of DNA replication ........................................................................................... 14 2.4.2 Replicative stress .............................................................................................................. 16 2.4.3 Replicative stress in cancer ............................................................................................... 16 2.5 Histones are essential for proper DNA replication .................................................................... 18 2.5.1 Regulation of replication-dependent histones ................................................................... 18 2.6 R-loops ..................................................................................................................................... 20 2.7 Scope of the study .................................................................................................................... 21 3 Manuscript ................................................................................................................................... 24 3.1 Abstract .................................................................................................................................... 25 3.2 Highlights .................................................................................................................................. 25 3.3 Significance .............................................................................................................................. 25 TABLE OF CONTENTS 3.4 Introduction ............................................................................................................................... 26 3.5 Methods and materials ............................................................................................................. 28 3.6 Results...................................................................................................................................... 36 3.7 Discussion ................................................................................................................................ 42 3.8 Acknowledgements .................................................................................................................. 45 3.9 Author contributions.................................................................................................................. 45 3.10 Declaration of Interests ............................................................................................................. 45 3.11 Figures/ Figure Legends ........................................................................................................... 47 3.12 Supplementary Figures/ Supplementary Figure Legends ........................................................ 63 4 Discussion .................................................................................................................................... 82 4.1 Interplay between the ISR and DNA replication ....................................................................... 83 4.1.1 DNA replication is inhibited upon ISR stimulation ............................................................. 83 4.1.2 The ISR does not activate replicative stress signalling ..................................................... 84 4.2 Crosstalk between DNA replication and protein translation ..................................................... 85 4.2.1 The processes of DNA and protein synthesis are co-regulated ........................................ 85 4.2.2 DNA replication proteins have long half-lives .................................................................... 86 4.3 Histones: a limiting factor in DNA replication ............................................................................ 87 4.3.1 Continuous histone synthesis ensures proper DNA replication ........................................ 87 4.3.2 ISR depletes cells of histones ........................................................................................... 87 4.3.3 Histone overexpression restores DNA replication upon ISR ............................................. 89 4.4 R-loops accumulate with the ISR ............................................................................................. 90 4.4.1 The ISR blocks DNA replication through R-loops independent of ATF4 ........................... 90 4.4.2 R-loops are enriched upon histone depletion .................................................................... 92 4.4.3 R-loops formed upon ISR are not threats to genomic stability .......................................... 94 4.5 Therapeutic potential of ISR in cancer ..................................................................................... 95 4.5.1 Activating the ISR to block DNA replication and proliferation in cancer ............................ 95 4.5.2 Inhibiting the ISR to suppress tumourigenesis .................................................................. 97 TABLE OF CONTENTS 4.5.3 Modulating the ISR in combination with other therapies ................................................... 99 4.6 Conclusions and future perspectives ...................................................................................... 100 5 References ................................................................................................................................. 102 6 Appendix .................................................................................................................................... 117 7 Abbreviations ............................................................................................................................. 121 8 List of Figures ............................................................................................................................ 127 9 Acknowledgements .................................................................................................................... 129 10 Curriculum
Load more

Recommended publications
  • 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]
  • Dna Replication in Archaea: Priming, Transferase, and Elongation Activities
    DNA REPLICATION IN ARCHAEA: PRIMING, TRANSFERASE, AND ELONGATION ACTIVITIES by Zhongfeng Zuo Bachelor degree, Beijing Technology and Business University, 1999 Submitted to the Graduate Faculty of the Kenneth P. Dietrich School of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2012 UNIVERSITY OF PITTSBURGH THE KENNETH P. DIETRICH SCHOOL OF ARTS AND SCIENCES This dissertation was presented by Zhongfeng Zuo It was defended on January 27, 2012 and approved by Stephen G. Weber, Professor, Department of Chemistry Billy Day, Professor, Department of Chemistry, Department of Pharmacy Renã A. S. Robinson, Assistant Professor, Department of Chemistry Dissertation Advisor: Michael A. Trakselis, Assistant Professor, Department of Chemistry ii DNA REPLICATION IN ARCHAEA: PRIMING, TRANSFERASE, AND ELONGATION ACTIVITIES Zhongfeng Zuo, Ph.D University of Pittsburgh, 2012 Copyright © by Zhongfeng Zuo 2012 iii DNA REPLICATION IN ARCHAEA: PRIMING, TRANSFERASE, AND ELONGATION ACTIVITIES Zhongfeng Zuo, Ph.D University of Pittsburgh, 2012 We have biochemically characterized the bacterial-like DnaG primase contained within the hyperthermophilic crenarchaeon Sulfolobus solfataricus (Sso ) and compared in vitro priming kinetics with those of the eukaryotic-like primase (PriS&L) also found in Sso . Sso DnaG exhibited metal- and temperature-dependent profiles consistent with priming at high temperatures. The distribution of primer products for Sso DnaG was discrete but highly similar to the distribution of primer products produced by the homologous Escherichia coli DnaG. The predominant primer length was 13 bases, although less abundant products of varying sizes are also present. Sso DnaG was found to bind DNA cooperatively as a dimer with a moderate dissociation constant.
    [Show full text]
  • DNA Or RNA Priming of Bacteriophage G4 DNA Synthesis By
    Proc. Nati. Acad. Sci. USA Vol. 74, No. 7, pp. 2815-2819, July 1977 Biochemistry DNA or RNA priming of bacteriophage G4 DNA synthesis by Escherichia coli dnaG protein (ADP/DNA binding protein/bacteriophage ST-1 DNA/DNA replication) SUE WICKNER Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 Communicated by Jerard Hurwitz, May 6, 1977 ABSTRACT Escherichia coli dnaG protein is involved in wild-type and thermolabile) were purified by procedures in the initiation of DNA synthesis dependent on G4 or ST-1 sin- refs. 13, 14, 15, 16, 5, and 17, respectively. Assay conditions and gle-stranded phage DNAs [Bouche, J.-P., Zechel, K. & Kornberg, definitions of units for DNA polymerase III are in ref. A. (1975)1. Biol. Chem. 250,5995-60011. The reaction occurs by 3; those the following mechanism: dnaG protein binds to specific sites for the other proteins are in the above references. DNA EF I on the DNA in a reaction requiring E. coli DNA binding protein. and DNA EF III were purified by procedures to be published An oligonucleotide is synthesized in a reaction involving dnaG elsewhere using the assay described in ref. 4. By native poly- protein, DNA binding protein, and DNA. With G4 DNA this acrylamide gel electrophoresis (18), the DNA binding protein reaction requires ADP, dTTP (or UTP), and dGTP (or GTP). was 80% pure and the dnaG protein from wild-type cells was Elongation of the oligonucleotide can be catalyzed by DNA 30% polymerase II or III in combination with dnaZ protein and pure.
    [Show full text]
  • 6.Start.Stop.07.Ppt [Read-Only]
    Accessory factors summary 1. DNA polymerase can’t replicate a genome. Solution ATP? No single stranded template Helicase + The ss template is unstable SSB (RPA (euks)) - No primer Primase (+) No 3’-->5’ polymerase Replication fork Too slow and distributive SSB and sliding clamp - Sliding clamp can’t get on Clamp loader (γ/RFC) + Lagging strand contains RNA Pol I 5’-->3’ exo, RNAseH - Lagging strand is nicked DNA ligase + Helicase introduces + supercoils Topoisomerase II + and products tangled 2. DNA replication is fast and processive DNA polymerase holoenzyme 1 Maturation of Okazaki fragments Topoisomerases control chromosome topology Catenanes/knots Topos Relaxed/disentangled •Major therapeutic target - chemotherapeutics/antibacterials •Type II topos transport one DNA through another 2 Starting and stopping summary 1. DNA replication is controlled at the initiation step. 2. DNA replication starts at specific sites in E. coli and yeast. 3. In E. coli, DnaA recognizes OriC and promotes loading of the DnaB helicase by DnaC (helicase loader) 4. DnaA and DnaC reactions are coupled to ATP hydrolysis. 5. Bacterial chromosomes are circular, and termination occurs opposite OriC. 6. In E. coli, the helicase inhibitor protein, tus, binds 7 ter DNA sites to trap the replisome at the end. 7. Eukaryotic chromosomes are linear, and the chromosome ends cannot be replicated by the replisome. 8. Telomerase extends the leading strand at the end. 9. Telomerase is a ribonucleoprotein (RNP) with RNA (template) and reverse-transcriptase subunits. Isolating DNA sequences that mediate initiation 3 Different origin sequences in different organisms E. Coli (bacteria) OriC Yeast ARS (Autonomously Replicating Sequences) Metazoans ???? Initiation in prokaryotes and eukaryotes Bacteria Eukaryotes ORC + other proteins load MCM hexameric helicases MCM (helicase) + RPA (ssbp) Primase + DNA pol α PCNA:pol δ + RFC MCM (helicase) + RPA (ssbp) PCNA:pol δ + RFC (clamp loader) Primase + DNA pol α PCNA:pol δ + DNA ligase 4 Crystal structure of DnaA:ATP revealed mechanism of origin assembly 1.
    [Show full text]
  • DNA Replication
    Semiconservative Replication DNA Replication Chapter 11 Wikipedia Kornberg’s DNA Polymerase I DNA Polymerase Reaction Requirements for DNA synthesis DNA Polymerase Mg++ dNTP single stranded DNA template Priming 3’ OH Consider how the active site descriminates among the dNTP’s. Other Enzymatic Activities Origin of Replication E. coli DNA Polymerases 9 mers- TTATTTCCAC Enzymatic Activities I II III . 5’-3’ synthesis + + + Pair 13mers GATCTCTTATTAG Required Primer + + + Order of Interaction 1. dnaA binds 9mers 3’-5’ Exonuclease + + + 2. dnaBC bind dnaA and 13mer -dnaB ATP dependent (Proofreading Activity) helicase 3. ssbp bind unwound DNA stabilizing it 5’-3’ Exonuclease 4. Gyrase (topoisomerase) relieves torsional stress (Replacement Activity) + - - . 5. Primase (dnaG) synthesizes short primers 6. DNA polymerase III initiates synthesis DNA Polymerase III mutation lethal – essential to replication DNA Polymerase I mutation viable – higher rate of mutation DNA Polymerase II mutation viable – function unknown 1 dnaA Discontinuous Replication DNA Polymerase synthesizes 5’-3’ only http://oregonstate.edu/instruction/bb492/figletters/FigH3.html Two Replication Forks Replication Fork http://oregonstate.edu/instruction/bb492/figletters/FigH2.html DNA Polymerase Clamp Replication Fork Improved Processivity http://www.wehi.edu.au/education/wehi-tv/dna/replication.html 2 Simultaneous Synthesis Eukaryotic Issues • Size of Genome • Ends of Linear Chromosomes Movie Size of Eukaryotic Genome Multiple Origins of Replication Genome Size – E coli 4.6 Mbp
    [Show full text]
  • Analysis of DNA Polymerases II and III in Mutants of Escherichia Coli Thermosensitive for DNA Synthesis (Polal Mutants/Phosphocellulose Chromatography/Dnae Locus)
    Proc. Nat. Acad. Sci. USA Vol. 68, No. 12, pp. 3150-3153, December 1971 Analysis of DNA Polymerases II and III in Mutants of Escherichia coli Thermosensitive for DNA Synthesis (polAl mutants/phosphocellulose chromatography/dnaE locus) MALCOLM L. GEFTER, YUKINORI HIROTA*, THOMAS KORNBERG, JAMES A. WECHSLER, AND C. BARNOUX* Department of Biological Sciences, Columbia University, New York, N.Y. 10027; and * Service de G6n6tique Cellulaire de l'Institut Pasteur, Paris Communicated by Cyrus Levinthal, October 18, 1971 ABSTRACT A series of double mutants carrying one of (5) PC79: F- his- strr malA xyl- mtlh thi- polAl sup- the thermosensitive mutations for DNA synthesis (dnaA, dnaD7 B, C, D, E, F, and G) and the polAl mutation of DeLucia and Cairns, were constructed. Enzyme activities of DNA (6) E5111: F- his- strr malA xylh mtlh arg- thi- sup- Polymerases II and III were measured in each mutant. polAl dnaE511 DNA Polymerase II activity was normal in all strains (7) E4860: F- his- str' malA xylh mtlh arg- thi- sup- tested. DNA Polymerase III activity is thermosensitive dnaE486 specifically in those strains having thermosensitive muta- tions at the dnaE locus. From these results we conclude (8) E4868: F- his- strr malA xyl- mtlh arg thi- sup- that DNA Polymerases II and III are independent en- polAl dnaE486 zymes and that DNA Polymerase III is an enzyme required (9) BT1026: H560thy-endoI- polAl dnaE for DNA replication in Escherichia coli. (10) BT1040: H560 thy endoI polAl dnaE (11) E1011: F- his- strr malA xyl- mtl- arg- thi- sup- The isolation by DeLucia and Cairns (1) of an Escherichia polAl dnaFI01 coli mutant that lacks DNA Polymerase I activity (polA1) (12) JW207: thy-rha-strrpolAl dnaF101 has prompted many investigations into the nature of the (13) NY73: leu- thy- metE rifr strr polAl dnaG3 DNA synthetic capacity of such strains.
    [Show full text]
  • Rnase HI Is Essential for Survival of Mycobacterium Smegmatis
    RESEARCH ARTICLE RNase HI Is Essential for Survival of Mycobacterium smegmatis Alina E. Minias1*, Anna M. Brzostek1, Malgorzata Korycka- Machala1, Bozena Dziadek2, Piotr Minias3, Malini Rajagopalan4, Murty Madiraju4, Jaroslaw Dziadek1* 1 Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland, 2 Department of Immunoparasitology, University of Lodz, Lodz, Poland, 3 Department of Teacher Training and Biodiversity Studies, University of Lodz, Lodz, Poland, 4 Department of Microbiology, University of Texas Health Center at Tyler, Tyler, Texas, United States of America * [email protected] (AM); [email protected] (JD) Abstract RNases H are involved in the removal of RNA from RNA/DNA hybrids. Type I RNases H are thought to recognize and cleave the RNA/DNA duplex when at least four ribonucleo- tides are present. Here we investigated the importance of RNase H type I encoding genes OPEN ACCESS for model organism Mycobacterium smegmatis. By performing gene replacement through homologous recombination, we demonstrate that each of the two presumable RNase H Citation: Minias AE, Brzostek AM, Korycka- Machala M, Dziadek B, Minias P, Rajagopalan M, et al. (2015) type I encoding genes, rnhA and MSMEG4305, can be removed from M. smegmatis ge- RNase HI Is Essential for Survival of Mycobacterium nome without affecting the growth rate of the mutant. Further, we demonstrate that deletion smegmatis. PLoS ONE 10(5): e0126260. of both RNases H type I encoding genes in M. smegmatis leads to synthetic lethality. Final- doi:10.1371/journal.pone.0126260 ly, we question the possibility of existence of RNase HI related alternative mode of initiation Academic Editor: Anil Kumar Tyagi, University of of DNA replication in M.
    [Show full text]
  • Mechanisms of Theta Plasmid Replication in Enterobacteria and Implications for Adaptation to Its Host JAY W
    DOMAIN 7 GENETICS AND GENETIC TOOLS Mechanisms of Theta Plasmid Replication in Enterobacteria and Implications for Adaptation to Its Host JAY W. KIM, VEGA BUGATA, GERARDO CORTÉS-CORTÉS, GISELLE QUEVEDO-MARTÍNEZ AND MANEL CAMPS Department of Microbiology and Environmental Toxicology, University of California at Santa Cruz, Santa Cruz, CA, 95064 ABSTRACT Plasmids are autonomously replicating sequences that help cells adapt to diverse stresses. Theta plasmids are the most frequent plasmid class in enterobacteria. They co-opt two host replication mechanisms: replication at oriC, a DnaA-dependent pathway leading to replisome assembly (theta class A), and replication fork restart, a PriA- dependent pathway leading to primosome assembly through primer extension and D-loop formation (theta classes B, C, and D). To ensure autonomy from the host’s replication and to facilitate copy number regulation, theta plasmids have unique mechanisms of replication initiation at the plasmid origin of replication (ori). Tight plasmid copy number regulation is Received: 26 March 2020 essential because of the major and direct impact plasmid gene dosage has on gene Accepted: 07 October 2020 expression. The timing of plasmid replication and segregation are also critical for opti- Posted: 18 November 2020 mizing plasmid gene expression. Therefore, we propose that plasmid replication needs to Editor: James M. Slauch, The School of be understood in its biological context, where complex origins of replication (redundant Molecular and Cellular Biology, University of origins, mosaic and cointegrated replicons), plasmid segregation, and toxin-antitoxin sys- Illinois at Urbana-Champaign, Urbana, IL; Gregory ori Phillips, College of Veterinary Medicine, Iowa tems are often present. Highlighting their tight functional integration with function, we State University, Ames, IA show that both partition and toxin-antitoxin systems tend to be encoded in close physical Citation: EcoSal Plus 2020; doi:10.1128/ proximity to the ori in a large collection of Escherichia coli plasmids.
    [Show full text]
  • Hexameric Helicase G40P Unwinds DNA in Single Base Pair Steps Michael Schlierf1,2*, Ganggang Wang3, Xiaojiang S Chen3, Taekjip Ha1,4,5,6,7*
    RESEARCH ARTICLE Hexameric helicase G40P unwinds DNA in single base pair steps Michael Schlierf1,2*, Ganggang Wang3, Xiaojiang S Chen3, Taekjip Ha1,4,5,6,7* 1Physics Department and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Illinois, United States; 2B CUBE – Center for Molecular Bioengineering, Technische Universita¨ t Dresden, Dresden, Germany; 3Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, United States; 4Howard Hughes Medical Institute, Baltimore, United States; 5Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, United States; 6Department of Biomedical Engineering, Johns Hopkins University, Baltimore, United States; 7Department of Biophysics, Johns Hopkins University, Baltimore, United States Abstract Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPgS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.
    [Show full text]
  • Antimycobacterial Activity of DNA Intercalator Inhibitors of Mycobacterium Tuberculosis Primase Dnag
    HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author J Antibiot Manuscript Author (Tokyo). Author Manuscript Author manuscript; available in PMC 2017 November 15. Published in final edited form as: J Antibiot (Tokyo). 2015 March ; 68(3): 153–157. doi:10.1038/ja.2014.131. Antimycobacterial activity of DNA intercalator inhibitors of Mycobacterium tuberculosis primase DnaG Chathurada Gajadeeraa,#, Melisa J. Willbyb,#, Keith D. Greena, Pazit Shaulc, Micha Fridmanc, Sylvie Garneau-Tsodikovaa,*, James E. Poseyb,*, and Oleg V. Tsodikova,* aDepartment of Pharmaceutical Sciences, University of Kentucky, Lexington, KY, 40536-0596, USA bDivision of Tuberculosis Elimination, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, GA, USA cSchool of Chemistry, Tel Aviv University, Tel Aviv, 66978, Israel Abstract Due to the rise in drug resistance in tuberculosis combined with the global spread of its causative pathogen, Mycobacterium tuberculosis (Mtb), innovative anti-mycobacterial agents are urgently needed. Recently, we developed a novel primase-pyrophosphatase assay and used it to discover inhibitors of an essential Mtb enzyme, primase DnaG (Mtb DnaG), a promising and unexplored potential target for novel anti-tuberculosis chemotherapeutics. Doxorubicin, an anthracycline antibiotic used as an anticancer drug, was found to be a potent inhibitor of Mtb DnaG. In this study, we investigated both inhibition of Mtb DnaG and the inhibitory activity against in vitro growth of Mtb and M. smegmatis (Msm) by other anthracyclines, daunorubicin and idarubicin, as well as by less cytotoxic DNA intercalators: aloe-emodin, rhein, and a mitoxantrone derivative. Generally, low-μM inhibition of Mtb DnaG by the anthracyclines was correlated with their low- μM minimum inhibitory concentrations.
    [Show full text]
  • Helicase Loader Complex Reveals Insights Into the Mechanism of Bacterial Primosome Assembly
    ARTICLE Received 20 Jun 2013 | Accepted 22 Aug 2013 | Published 19 Sep 2013 DOI: 10.1038/ncomms3495 OPEN Structure of a helicase–helicase loader complex reveals insights into the mechanism of bacterial primosome assembly Bin Liu1,2, William K. Eliason1,2 & Thomas A. Steitz1,2,3 During the assembly of the bacterial loader-dependent primosome, helicase loader proteins bind to the hexameric helicase ring, deliver it onto the oriC DNA and then dissociate from the complex. Here, to provide a better understanding of this key process, we report the crystal structure of the B570-kDa prepriming complex between the Bacillus subtilis loader protein and the Bacillus stearothermophilus helicase, as well as the helicase-binding domain of primase with a molar ratio of 6:6:3 at 7.5 Å resolution. The overall architecture of the complex exhibits a three-layered ring conformation. Moreover, the structure combined with the proposed model suggests that the shift from the ‘open-ring’ to the ‘open-spiral’ and then the ‘closed- spiral’ state of the helicase ring due to the binding of single-stranded DNA may be the cause of the loader release. 1 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA. 2 Howard Hughes Medical Institute, New Haven, Connecticut 06510, USA. 3 Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA. Correspondence and requests for materials should be addressed to T.A.S. (email: [email protected]). NATURE COMMUNICATIONS | 4:2495 | DOI: 10.1038/ncomms3495 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited. All rights reserved.
    [Show full text]
  • Architecture and Conservation of the Bacterial DNA Replication Machinery, an Underexploited Drug Target
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Research Online University of Wollongong Research Online Faculty of Science - Papers (Archive) Faculty of Science, Medicine and Health 2012 Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target Andrew Robinson University of Wollongong, [email protected] Rebecca J. Causer University of Wollongong, [email protected] Nicholas E. Dixon University of Wollongong, [email protected] Follow this and additional works at: https://ro.uow.edu.au/scipapers Part of the Life Sciences Commons, Physical Sciences and Mathematics Commons, and the Social and Behavioral Sciences Commons Recommended Citation Robinson, Andrew; Causer, Rebecca J.; and Dixon, Nicholas E.: Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target 2012, 352-372. https://ro.uow.edu.au/scipapers/2996 Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: [email protected] Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target Abstract "New antibiotics with novel modes of action are required to combat the growing threat posed by multi- drug resistant bacteria. Over the last decade, genome sequencing and other high-throughput techniques have provided tremendous insight into the molecular processes underlying cellular functions in a wide range of bacterial species. We can now use these data to assess the degree of conservation of certain aspects of bacterial physiology, to help choose the best cellular targets for development of new broad- spectrum antibacterials.
    [Show full text]