DOCSLIB.ORG

Mechanisms of Theta Plasmid Replication in Enterobacteria and Implications for Adaptation to Its Host JAY W

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read 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. We also propose that ecosalplus.ESP-0026-2019. adaptation of plasmids to their host optimizes their contribution to the host’s fitness while Correspondence: Manel Camps, mcamps@ restricting access to broad genetic diversity, and we argue that this trade-off between ucsc.edu adaptation to host and access to genetic diversity is likely a determinant factor shaping Copyright: © 2020 American Society for the distribution of replicons in populations of enterobacteria. Microbiology. All rights reserved. doi:10.1128/ecosalplus.ESP-0026-2019 INTRODUCTION Plasmids are autonomously replicating sequences. They are found across all kingdoms of life but are particularly abundant in prokaryotes (1). They typically carry nonessential genes that have adaptive value under specific stresses such as heavy metal exposure, antibiotics, or adapting to a host (virulence genes) (2, 3). Here, we review the mechanism of plasmid replication known as theta, which is by far the predominant type in Enterobacteriaceae. This prokaryotic family groups a number of related genera (Escherichia, Shigella, Salmonella, Yersinia) that share the intestinal tract as their central ecological niche and that have been extensively studied due to their clinical relevance (4, 5). ASMScience.org/EcoSalPlus 1 Downloaded from www.asmscience.org by IP: 128.114.255.203 On: Wed, 18 Nov 2020 19:01:02 Kim et al. Enterobacterial plasmids share similar features. Nearly all restricted to replication initiation, with downstream steps of them can be transferred between strains in one way or frequently relying exclusively on host proteins. Having another, a process known as horizontal gene transfer plasmid-specific mechanisms of replication initiation (HGT). This allows plasmids to serve as platforms for the ensures autonomy from the host and facilitates the reg- exchange of genetic material, for capturing new adaptive ulation of plasmid copy number by providing specific genes from the environmental microbial gene pool, and targets for regulation. Plasmid replication initiation for shuffling and spreading these genes among pathogens involves plasmid-encoded replication proteins (Reps) (3, 6, 7). There are three main mechanisms of HGT, and/or plasmid-specific primers. Even these plasmid namely, cell-to-cell contact and formation of a bridge replication initiation factors are often assisted by host (conjugation), direct uptake from the environment proteins (see below). (transformation), and through bacteriophages (transduc- tion) (8). Conjugation is the predominant mechanism in Host proteins participating in plasmid DNA synthesis are Gram-negative bacteria. It involves copying the plasmid part of the machinery that replicates the chromosome at DNA, relaxing it, and moving the DNA through a type IV oriC (recently reviewed in references 8 and 13–15) and/or secretion system to a recipient cell in an ATP-dependent that restart replication following replication fork collapse fashion (reviewed in references 3, 7,and9). The majority (reviewed in references 16–19). These include proteins of enterobacterial plasmids carry sets of conjugation facilitating the melting of the DNA duplex (DnaA), genes, although these are not always active or complete helicases (PriA, DnaB), proteins that catalyze primer (10). Some virulence plasmids do not have a conjugation synthesis (DnaG) and primer extension (Pol I, Pol III machinery but can be transferred along with a conjugative holoenzyme), proteins that process primers and gaps plasmid, which acts as a helper; this process is known as (Pol I, ligase), proteins that control the local supercoiling plasmid mobilization (10). Transduction is also frequent status of DNA (topoisomerases), and proteins that bind in enterobacteria (11). It occurs when a bacteriophage and stabilize single-stranded DNA (ssDNA) (SSB). Note packages bacterial genetic material along with its own that replication proceeds in the 5′ to 3′ direction. The genetic material and infects another bacterium (general- synchronous replication of leading and lagging strands ized transduction) or when a phage that has integrated involves two core Pol III enzymes, with the enzyme into the bacterial chromosome is improperly excised and replicating the leading strand doing so continuously and packages bacterial DNA (specialized transduction) (12). the enzyme replicating the lagging strand doing so dis- continuously, i.e., in segments. The Pol III core enzymes Here, we present our current mechanistic understanding in the Pol III holoenzyme are joined by two τ (tau) of theta plasmid replication and its regulation. We also subunits and assisted by β units that act as mobile sliding present concepts that are emerging from genomic stud- clamps, which ensures the coordination and processivity ies, which help place the replication of these plasmids in of the replication in the two strands. its broader biological context. Given that plasmids iso- lated from a variety of Enterobacteriaceae share similar features (5), we use virulence plasmids in extraintestinal MECHANISMS OF PLASMID REPLICATION pathogenic Escherichia coli (ExPEC) as representative of Replication initiation determines the mechanics of plas- enterobacteria. mid replication. Within theta plasmids, we can distinguish two broad mechanisms of replication initiation: oriC-like (which parallels replication initiation at oriC)andPriA- PLASMID REPLICATION INITIATION AT ORIGINS mediated (which is similar to replication restart after OF REPLICATION replication fork collapse). The mechanisms involved in Plasmid replication starts at a specific location known as these two categories of plasmid replication (as well as those the plasmid origin of replication, or ori. The origin of of nontheta plasmids) are systematically compared in replication contains nucleotide sequence motifs that are Table 1 and discussed in detail below. recognized in trans by the replication machinery. This machinery typically involves a combination of plasmid oriC-like plasmids correspond to class A theta plasmid and host elements. Since borrowing genes from the replication. PriA-mediated plasmids correspond to theta host minimizes the metabolic cost of plasmid mainte- classes B, C, and D. Both categories converge at the DnaB nance, plasmid-encoded replicative elements are typically loading step and rely on the hosts’ Pol III holoenzyme to 2 ASMScience.org/EcoSalPlus Downloaded from www.asmscience.org by IP: 128.114.255.203 On: Wed, 18 Nov 2020 19:01:02 ASMScience.org/EcoSalPlus Table 1 Comparison of the different modes of plasmid replicationa Characteristic Theta A Theta Bb Theta C Theta D Strand Rolling-circle displacement Directionality of Bi/uni Uni Uni Uni Bi Uni replication (unidirectional vs bi-directional) Coupling of leading- Coupled Coupled Coupled Coupled Uncoupled Uncoupled and lagging-strand (replisome) (replisome) (replisome) (replisome) synthesis Simultaneous Yes Yes Yes Yes Yes No, with generation of leading- and lagging- single-stranded inter- strand synthesis mediate Leading strand synthesis Continuous vs Continuous Continuous Continuous Continuous Continuous Continuous discontinuous Initiation factor Rep melts duplex NA RepA (also primase Rep E melts duplex only Rep C (DnaA-like) Rep (generates initial and recruits DnaB activity) and has active role in nick and seals it) helicase, primer generation often assisted by DnaA and by NAPs Initiation factor Multiple iterons NA Iteron with two functional No iterons, no bending ssi sites
Load more

Recommended publications
  • Replisome Assembly at Oric, the Replication Origin of E. Coli, Reveals an Explanation for Initiation Sites Outside an Origin
    Molecular Cell, Vol. 4, 541±553, October, 1999, Copyright 1999 by Cell Press Replisome Assembly at oriC, the Replication Origin of E. coli, Reveals an Explanation for Initiation Sites outside an Origin Linhua Fang,*§ Megan J. Davey,² and Mike O'Donnell²³ have not been addressed. For example, is the local un- *Microbiology Department winding sufficiently large for two helicases to assemble Joan and Sanford I. Weill Graduate School of Medical for bidirectional replication, or does one helicase need Sciences of Cornell University to enter first and expand the bubble via helicase action New York, New York 10021 to make room for the second helicase? The known rep- ² The Rockefeller University and licative helicases are hexameric and encircle ssDNA. Howard Hughes Medical Institute Which strand does the initial helicase(s) at the origin New York, New York 10021 encircle, and if there are two, how are they positioned relative to one another? Primases generally require at least transient interaction with helicase to function. Can Summary primase function with the helicase(s) directly after heli- case assembly at the origin, or must helicase-catalyzed This study outlines the events downstream of origin DNA unwinding occur prior to RNA primer synthesis? unwinding by DnaA, leading to assembly of two repli- Chromosomal replicases are comprised of a ring-shaped cation forks at the E. coli origin, oriC. We show that protein clamp that encircles DNA, a clamp-loading com- two hexamers of DnaB assemble onto the opposing plex that uses ATP to assemble the clamp around DNA, strands of the resulting bubble, expanding it further, and a DNA polymerase that binds the circular clamp, yet helicase action is not required.
    [Show full text]
  • USP7 Couples DNA Replication Termination to Mitotic Entry
    bioRxiv preprint doi: https://doi.org/10.1101/305318; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. USP7 couples DNA replication termination to mitotic entry Antonio Galarreta1*, Emilio Lecona1*, Pablo Valledor1, Patricia Ubieto1,2, Vanesa Lafarga1, Julia Specks1 & Oscar Fernandez-Capetillo1,3 1Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain 2Current Address: DNA Replication Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain 3Science for Life Laboratory, Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 21 Stockholm, Sweden *Co-first authors Correspondence: E.L. ([email protected]) or O.F. ([email protected]) Lead Contact: Oscar Fernandez-Capetillo Spanish National Cancer Research Centre (CNIO) Melchor Fernandez Almagro, 3 Madrid 28029, Spain Tel.: +34.91.732.8000 Ext: 3480 Fax: +34.91.732.8028 Email: [email protected] KEYWORDS: USP7; CDK1; DNA REPLICATION; MITOSIS; S/M TRANSITION. bioRxiv preprint doi: https://doi.org/10.1101/305318; this version posted April 20, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. USP7 coordinates the S/M transition 2 SUMMARY To ensure a faithful segregation of chromosomes, DNA must be fully replicated before mitotic entry. However, how cells sense the completion of DNA replication and to what extent this is linked to the activation of the mitotic machinery remains poorly understood. We previously showed that USP7 is a replisome-associated deubiquitinase with an essential role in DNA replication.
    [Show full text]
  • Arthur Kornberg Discovered (The First) DNA Polymerase Four
    Arthur Kornberg discovered (the first) DNA polymerase Using an “in vitro” system for DNA polymerase activity: 1. Grow E. coli 2. Break open cells 3. Prepare soluble extract 4. Fractionate extract to resolve different proteins from each other; repeat; repeat 5. Search for DNA polymerase activity using an biochemical assay: incorporate radioactive building blocks into DNA chains Four requirements of DNA-templated (DNA-dependent) DNA polymerases • single-stranded template • deoxyribonucleotides with 5’ triphosphate (dNTPs) • magnesium ions • annealed primer with 3’ OH Synthesis ONLY occurs in the 5’-3’ direction Fig 4-1 E. coli DNA polymerase I 5’-3’ polymerase activity Primer has a 3’-OH Incoming dNTP has a 5’ triphosphate Pyrophosphate (PP) is lost when dNMP adds to the chain E. coli DNA polymerase I: 3 separable enzyme activities in 3 protein domains 5’-3’ polymerase + 3’-5’ exonuclease = Klenow fragment N C 5’-3’ exonuclease Fig 4-3 E. coli DNA polymerase I 3’-5’ exonuclease Opposite polarity compared to polymerase: polymerase activity must stop to allow 3’-5’ exonuclease activity No dNTP can be re-made in reversed 3’-5’ direction: dNMP released by hydrolysis of phosphodiester backboneFig 4-4 Proof-reading (editing) of misincorporated 3’ dNMP by the 3’-5’ exonuclease Fidelity is accuracy of template-cognate dNTP selection. It depends on the polymerase active site structure and the balance of competing polymerase and exonuclease activities. A mismatch disfavors extension and favors the exonuclease.Fig 4-5 Superimposed structure of the Klenow fragment of DNA pol I with two different DNAs “Fingers” “Thumb” “Palm” red/orange helix: 3’ in red is elongating blue/cyan helix: 3’ in blue is getting edited Fig 4-6 E.
    [Show full text]
  • Polymerase Δ Deficiency Causes Syndromic Immunodeficiency with Replicative Stress
    Polymerase δ deficiency causes syndromic immunodeficiency with replicative stress Cecilia Domínguez Conde, … , Mirjam van der Burg, Kaan Boztug J Clin Invest. 2019. https://doi.org/10.1172/JCI128903. Research Article Genetics Immunology Graphical abstract Find the latest version: https://jci.me/128903/pdf The Journal of Clinical Investigation RESEARCH ARTICLE Polymerase δ deficiency causes syndromic immunodeficiency with replicative stress Cecilia Domínguez Conde,1,2 Özlem Yüce Petronczki,1,2,3 Safa Baris,4,5 Katharina L. Willmann,1,2 Enrico Girardi,2 Elisabeth Salzer,1,2,3,6 Stefan Weitzer,7 Rico Chandra Ardy,1,2,3 Ana Krolo,1,2,3 Hanna Ijspeert,8 Ayca Kiykim,4,5 Elif Karakoc-Aydiner,4,5 Elisabeth Förster-Waldl,9 Leo Kager,6 Winfried F. Pickl,10 Giulio Superti-Furga,2,11 Javier Martínez,7 Joanna I. Loizou,2 Ahmet Ozen,4,5 Mirjam van der Burg,8 and Kaan Boztug1,2,3,6 1Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, 2CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, and 3St. Anna Children’s Cancer Research Institute (CCRI), Vienna, Austria. 4Pediatric Allergy and Immunology, Marmara University, Faculty of Medicine, Istanbul, Turkey. 5Jeffrey Modell Diagnostic Center for Primary Immunodeficiency Diseases, Marmara University, Istanbul, Turkey. 6St. Anna Children’s Hospital, Department of Pediatrics and Adolescent Medicine, Vienna, Austria. 7Center for Medical Biochemistry, Medical University of Vienna, Vienna, Austria. 8Department of Pediatrics, Laboratory for Immunology, Leiden University Medical Centre, Leiden, Netherlands. 9Department of Neonatology, Pediatric Intensive Care and Neuropediatrics, Department of Pediatrics and Adolescent Medicine, 10Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, and 11Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.
    [Show full text]
  • The Obscure World of Integrative and Mobilizable Elements Gérard Guédon, Virginie Libante, Charles Coluzzi, Sophie Payot-Lacroix, Nathalie Leblond-Bourget
    The obscure world of integrative and mobilizable elements Gérard Guédon, Virginie Libante, Charles Coluzzi, Sophie Payot-Lacroix, Nathalie Leblond-Bourget To cite this version: Gérard Guédon, Virginie Libante, Charles Coluzzi, Sophie Payot-Lacroix, Nathalie Leblond-Bourget. The obscure world of integrative and mobilizable elements: Highly widespread elements that pirate bacterial conjugative systems. Genes, MDPI, 2017, 8 (11), pp.337. 10.3390/genes8110337. hal- 01686871 HAL Id: hal-01686871 https://hal.archives-ouvertes.fr/hal-01686871 Submitted on 26 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License G C A T T A C G G C A T genes Review The Obscure World of Integrative and Mobilizable Elements, Highly Widespread Elements that Pirate Bacterial Conjugative Systems Gérard Guédon *, Virginie Libante, Charles Coluzzi, Sophie Payot and Nathalie Leblond-Bourget * ID DynAMic, Université de Lorraine, INRA, 54506 Vandœuvre-lès-Nancy, France; [email protected] (V.L.); [email protected] (C.C.); [email protected] (S.P.) * Correspondence: [email protected] (G.G.); [email protected] (N.L.-B.); Tel.: +33-037-274-5142 (G.G.); +33-037-274-5146 (N.L.-B.) Received: 12 October 2017; Accepted: 15 November 2017; Published: 22 November 2017 Abstract: Conjugation is a key mechanism of bacterial evolution that involves mobile genetic elements.
    [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]
  • DNA Polymerases at the Eukaryotic Replication Fork Thirty Years After: Connection to Cancer
    cancers Review DNA Polymerases at the Eukaryotic Replication Fork Thirty Years after: Connection to Cancer Youri I. Pavlov 1,2,* , Anna S. Zhuk 3 and Elena I. Stepchenkova 2,4 1 Eppley Institute for Research in Cancer and Allied Diseases and Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA 2 Department of Genetics and Biotechnology, Saint-Petersburg State University, 199034 Saint Petersburg, Russia; [email protected] 3 International Laboratory of Computer Technologies, ITMO University, 197101 Saint Petersburg, Russia; [email protected] 4 Laboratory of Mutagenesis and Genetic Toxicology, Vavilov Institute of General Genetics, Saint-Petersburg Branch, Russian Academy of Sciences, 199034 Saint Petersburg, Russia * Correspondence: [email protected] Received: 30 September 2020; Accepted: 13 November 2020; Published: 24 November 2020 Simple Summary: The etiology of cancer is linked to the occurrence of mutations during the reduplication of genetic material. Mutations leading to low replication fidelity are the culprits of many hereditary and sporadic cancers. The archetype of the current model of replication fork was proposed 30 years ago. In the sequel to our 2010 review with the words “years after” in the title inspired by A. Dumas’s novels, we go over new developments in the DNA replication field and analyze how they help elucidate the effects of the genetic variants of DNA polymerases on cancer. Abstract: Recent studies on tumor genomes revealed that mutations in genes of replicative DNA polymerases cause a predisposition for cancer by increasing genome instability. The past 10 years have uncovered exciting details about the structure and function of replicative DNA polymerases and the replication fork organization.
    [Show 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,
    [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]
  • Plasmid Replication-Associated Single-Strand-Specific
    12858–12873 Nucleic Acids Research, 2020, Vol. 48, No. 22 Published online 3 December 2020 doi: 10.1093/nar/gkaa1163 Plasmid replication-associated single-strand-specific methyltransferases Alexey Fomenkov 1,*, Zhiyi Sun1, Iain A. Murray 1, Cristian Ruse1, Colleen McClung1, Yoshiharu Yamaichi 2, Elisabeth A. Raleigh 1,* and Richard J. Roberts1,* 1New England Biolabs Inc., 240 County Road, Ipswich, MA, USA and 2Universite´ Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Downloaded from https://academic.oup.com/nar/article/48/22/12858/6018438 by guest on 24 September 2021 Received August 06, 2020; Revised November 10, 2020; Editorial Decision November 11, 2020; Accepted November 12, 2020 ABSTRACT RM-associated modification and the diversity of associ- ated functions remains incompletely understood (3). Long- Analysis of genomic DNA from pathogenic strains read, modification-sensitive SMRT sequencing technology of Burkholderia cenocepacia J2315 and Escherichia has facilitated sequencing and assembly of a wide variety coli O104:H4 revealed the presence of two unusual of genomes and also clarified the modification repertoire. MTase genes. Both are plasmid-borne ORFs, carried Detection of the modification status of bases is possible by pBCA072 for B. cenocepacia J2315 and pESBL for for m6A, m4C and oxidized forms of m5C modified bases E. coli O104:H4. Pacific Biosciences SMRT sequenc- (m5hC and 5caC) (4). Recently, high throughput analy- ing was used to investigate DNA methyltransferases sis of 230 diverse bacterial and archaeal methylomes strik- M.BceJIII and M.EcoGIX, using artificial constructs. ingly revealed that almost 50% of organisms harbor Type Mating properties of engineered pESBL derivatives II DNA methyltransferases (MTase) homologs with no ap- were also investigated.
    [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]