DOCSLIB.ORG

Downloaded from Received September 13, 2010; Revised March 17, 2011; Accepted April 4, 2011

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Downloaded from Received September 13, 2010; Revised March 17, 2011; Accepted April 4, 2011 http://www.ncbi.nlm.nih.gov/pubmed/21622956. Postprint available at: http://www.zora.uzh.ch Posted at the Zurich Open Repository and Archive, University of Zurich. University of Zurich http://www.zora.uzh.ch Zurich Open Repository and Archive Originally published at: Rossi, F; Khanduja, J S; Bortoluzzi, A; Houghton, J; Sander, P; Güthlein, C; Davis, E O; Springer, B; Böttger, E C; Relini, A; Penco, A; Muniyappa, K; Rizzi, M (2011). The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action. Nucleic Acids Winterthurerstr. 190 Research:Epub ahead of print. CH-8057 Zurich http://www.zora.uzh.ch Year: 2011 The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action Rossi, F; Khanduja, J S; Bortoluzzi, A; Houghton, J; Sander, P; Güthlein, C; Davis, E O; Springer, B; Böttger, E C; Relini, A; Penco, A; Muniyappa, K; Rizzi, M http://www.ncbi.nlm.nih.gov/pubmed/21622956. Postprint available at: http://www.zora.uzh.ch Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch Originally published at: Rossi, F; Khanduja, J S; Bortoluzzi, A; Houghton, J; Sander, P; Güthlein, C; Davis, E O; Springer, B; Böttger, E C; Relini, A; Penco, A; Muniyappa, K; Rizzi, M (2011). The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action. Nucleic Acids Research:Epub ahead of print. The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action Abstract Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis. Nucleic Acids Research Advance Access published May 27, 2011 Nucleic Acids Research, 2011, 1–13 doi:10.1093/nar/gkr271 The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action Franca Rossi1, Jasbeer Singh Khanduja2, Alessio Bortoluzzi1, Joanna Houghton3, Peter Sander4, Carolin Gu¨thlein4, Elaine O. Davis3, Burkhard Springer5, Erik C. Bo¨ ttger4, Annalisa Relini6, Amanda Penco6, K. Muniyappa2 and Menico Rizzi1,* 1DiSCAFF, University of Piemonte Orientale ‘Amedeo Avogadro’, Via Bovio 6, 28100 Novara, Italy, 2Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India, 3Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway Mill Hill, London, UK, 4Institut fu¨ r Medizinische Mikrobiologie, University of Zu¨ rich, Gloriastrasse 30/32, CH-8006 Zu¨ rich, Switzerland, 5Institut fu¨ r Medizinische Mikrobiologie und Hygiene, Universita¨ tsplatz 4, 8010 Graz, Austria and 6Department of Physics, University of Genoa, Via Dodecaneso, 33, 16146 Genoa, Italy Downloaded from Received September 13, 2010; Revised March 17, 2011; Accepted April 4, 2011 ABSTRACT tion of small-molecule inhibitors interfering with nar.oxfordjournals.org Mycobacterium tuberculosis is an extremely well the NER activity in M. tuberculosis. adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical INTRODUCTION at Universität Zürich. Hauptbibliothek Irchel on July 22, 2011 assaults originating from the host defence mechan- During its life, Mycobacterium tuberculosis is exposed to isms. As a consequence, this bacterium is thought numerous genotoxic insults originating from both anti- to possess highly efficient DNA repair machineries, microbial host defence mechanisms and the environment the nucleotide excision repair (NER) system (1). Since persistence within the infected macrophage and amongst these. Although NER is of central reactivation of the bacillus from the dormant state are importance to DNA repair in M. tuberculosis, our key features of infection, the maintenance of genome understanding of the processes in this species is integrity is considered a vital aspect in the biology of limited. The conserved UvrABC endonuclease M. tuberculosis (2,3). represents the multi-enzymatic core in bacterial One of the major molecular machines that control chromosome stability in living species is represented by NER, where the UvrA ATPase provides the DNA the nucleotide excision repair (NER) system (4–7) that is lesion-sensing function. The herein reported capable of repairing a wide variety of DNA lesions genetic analysis demonstrates that M. tuberculosis (reviewed in ref. 5). In Eubacteria and some Archaea, UvrA is important for the repair of nitrosative and the first steps in NER are carried out by UvrA, UvrB oxidative DNA damage. Moreover, our biochemical and UvrC proteins, often referred to as the UvrABC and structural characterization of recombinant endonuclease. This multi-enzymatic complex recognizes M. tuberculosis UvrA contributes new insights into the damage and excises a short lesion-containing DNA its mechanism of action. In particular, the structural oligonucleotide, in a multi-step process in which the investigation reveals an unprecedented conform- three proteins act in concert. Briefly, an UvrAB ation of the UvrB-binding domain that we propose heteromeric complex scans the DNA searching for to be of functional relevance. Taken together, damaged sites. The identity of the UvrAB oligomeric state and of the searching complex have long been our data suggest UvrA as a potential target for the the subject of debate and controversy; however, two inde- development of novel anti-tubercular agents and pendent studies using fluorescence resonance energy provide a biochemical framework for the identifica- *To whom correspondence should be addressed. Tel: +39 0321 375 712; Fax: +39 0321 375 821; Email: [email protected] The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. ß The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 Nucleic Acids Research, 2011 transfer and single-molecule imaging of quantum-dot- We report here an integrated analysis of M. tuberculosis labelled proteins have proposed that the ATP-bound UvrA. Our data indicate a key functional role for UvrA in UvrA2B2 complex is the stable form that slides along protection against reactive oxygen and nitrogen the DNA until a damaged site is encountered (8,9). species-triggered DNA damage. Moreover, we show that Once recognized by the UvrA component, the injured the DNA binding activity of M. tuberculosis UvrA towards strand is transferred to UvrB and UvrA is released. various DNA structures mimicking intermediates of DNA UvrC is then recruited at the UvrBDNA complex repair, is correlated to the amount of single-stranded where it performs a single strand incision on both sides regions within the DNA substrate and provide evidence of the damaged site. Subsequently, the helicase UvrD that the low intrinsic ATPase activity of M. tuberculosis removes the excised oligonucleotide and DNA polymerase UvrA is markedly stimulated by DNA. Finally, the crystal I fills the gap, using the intact complementary strand as structure determination of M. tuberculosis UvrA in its the template. Finally, DNA ligase seals the nick, thus ligand-free form reveals a remarkable repositioning of restoring the integrity of the double helix (5). the UvrB binding domain and could therefore represent Information is beginning to emerge concerning the role a further insight in the very initial steps of UvrABC of NER in the biology of mycobacteria, an observation complex assembling at the site of DNA lesion. that assumes even more emphasis considering that these bacteria lack a functional DNA mismatch repair system (2). Transcription of the uvrA gene has been shown to be MATERIALS AND METHODS up-regulated in human macrophage-grown M. tubercu- Functional characterization of M. tuberculosis uvrA Downloaded from losis bacilli hours post-infection (10). Gene inactivation mutant strain and trans-complementation analyses demonstrated a crucial role
Load more

Recommended publications
  • DNA Repair Systems Guardians of the Genome
    GENERAL ARTICLE DNA Repair Systems Guardians of the Genome D N Rao and Yedu Prasad The 2015 Nobel Prize in Chemistry was awarded jointly to Tomas Lindahl, Paul Modrich and Aziz Sancar to honour their accomplishments in the field of DNA repair. Ever since the discovery of DNA structure and their importance in the storage of genetic information, questions about their stability became pertinent. A molecule which is crucial for the de- velopment and propagation of an organism must be closely D N Rao is a professor at the monitored so that the genetic information is not corrupted. Department of Biochemistry, Thanks to the pioneering research work of Lindahl, Sancar, Indian Institute of Science, Modrich and their colleagues, we now have an holistic aware- Bengaluru. His research ness of how DNA damage occurs and how the damage is rec- work primarily focuses on DNA interacting proteins in tified in bacteria as well as in higher organisms including hu- prokaryotes. This includes man beings. A comprehensive understanding of DNA repair restriction-modification has proven crucial in the fight against cancer and other debil- systems, DNA repair proteins itating diseases. from pathogenic bacteria and and proteins involved in The genetic information that guides the development, metabolism horizontal gene transfer and and reproduction of all living organisms and many viruses resides DNA recombination. in the DNA. This biological information is stored in the DNA Yedu Prasad is a graduate molecule as combinations of sequences that are formed by purine student working on his PhD and pyrimidine bases attached to the deoxyribose sugar (Figure under the guidance of D N 1).
    [Show full text]
  • Base Excision Repair Synthesis of DNA Containing 8-Oxoguanine in Escherichia Coli
    EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 35, No. 2, 106-112, April 2003 Base excision repair synthesis of DNA containing 8-oxoguanine in Escherichia coli Yun-Song Lee1,3 and Myung-Hee Chung2 Introduction 1Division of Pharmacology 8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a muta- Department of Molecular and Cellular Biology genic adduct formed by reactive oxygen species Sungkyunkwan University School of Medicine (Kasai and Nishimura, 1984). As a structural prefe- Suwon 440-746, Korea rence, adenine is frequently incorporated into oppo- 2Department of Pharmacology site template 8-oxo-G (Shibutani et al., 1991), and 8- Seoul National University College of Medicine oxo-dGTP is incorporated into opposite template dA Jongno-gu, Seoul 110-799, Korea during DNA synthesis (Cheng et al., 1992). Thus, un- 3Corresponding author: Tel, 82-31-299-6190; repaired, these mismatches lead to GT and AC trans- Fax, 82-31-299-6209; E-mail, [email protected] versions, respectively (Grollman and Morya, 1993). In Escherichia coli, several DNA repair enzymes, Accepted 29 March 2003 preventing mutagenesis by 8-oxo-G, are known as the GO system (Michaels et al., 1992). The GO system Abbreviations: 8-oxo-G, 8-oxo-7,8-dihydroguanine; Fapy, 2,6-dihy- consists of MutT (8-oxo-dGTPase), MutM (2,6-dihydro- droxy-5N-formamidopyrimidine; FPG, Fapy-DNA glycosylase; BER, xy-5N-formamidopyrimidine (Fapy)-DNA glycosylase, base excision repair; AP, apurinic/apyrimidinic; dRPase, deoxyribo- Fpg) and MutY (adenine-DNA glycosylase). 8-oxo- phosphatase GTPase prevents incorporation of 8-oxo-dGTP into DNA by degrading 8-oxo-dGTP.
    [Show full text]
  • Uvrc Coordinates an O2-Sensitive [4Fe4s] Cofactor
    HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author J Am Chem Manuscript Author Soc. Author Manuscript Author manuscript; available in PMC 2020 July 30. Published in final edited form as: J Am Chem Soc. 2020 June 24; 142(25): 10964–10977. doi:10.1021/jacs.0c01671. UvrC Coordinates an O2-Sensitive [4Fe4S] Cofactor Rebekah M. B. Silva, Michael A. Grodick, Jacqueline K. Barton Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United States Abstract Recent advances have led to numerous landmark discoveries of [4Fe4S] clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV–visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with [4Fe4S] coordination with 60–70% cofactor incorporation, and additionally, we show that, bound to UvrC, the [4Fe4S] cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor.
    [Show full text]
  • Jiao Da Biochemistry-2
    DNA Damage and Repair Guo-Min Li (李国民 ）, Ph.D. Professor James Gardner Chair in Cancer Research University of Kentucky College of Medicine DNA Damage and Repair Contents • DNA damage • DNA damage response • DNA repair – Direct reversal – Base excision repair – Nucleotide excision repair – Translesion synthesis – Mismatch repair – Double strand break repair • Recombination repair • Non-homologous end joining – Inter strand cross-link repair Major DNA Lesions DNA lesions and structures that elicit DNA response reactions. Some of the base backbone lesions and noncanonical DNA structures that elicit DNA response reactions are shown. O6MeGua indicates O6- methyldeoxyguanosine, T<>T indicates a cyclobutane thymine dimer, and the cross-link shown is cisplatin G-G interstrand cross-link. Endogenous DNA Damage Deamination of cytosine to uracil can spontaneously occurs to creates a U·G mismatch. Depurination removes a base from DNA, blocking replication and transcription. Mismatch Derived from Normal DNA Metabolism 5’ 3’ DNA replication errors DNA recombination DNA Damage and Repair Contents • DNA damage • DNA damage response • DNA repair – Direct reversal – Base excision repair – Nucleotide excision repair – Translesion synthesis – Mismatch repair – Double strand break repair • Recombination repair • Non-homologous end joining – Inter strand cross-link repair Cellular Response to DNA Damage A contemporary view of the general outline of the DNA damage response signal-transduction pathway. Arrowheads represent activating events and perpendicular ends represent inhibitory events. Cell- cycle arrest is depicted with a stop sign, apoptosis with a tombstone. The DNA helix with an arrow represents damage-induced transcription, while the DNA helix with several oval-shaped subunits represents damage-induced repair. For the purpose of simplicity, the network of interacting pathways are depicted as a linear pathway consisting of signals, sensors, transducers and effectors.
    [Show full text]
  • Interactions of the UVRABC Endonuclease in Vivo and in Vitro with DMA Damage Produced by Antineoplastic Anthracyclines1
    [CANCER RESEARCH 44, 3489-3492, August 1984] Vii -. Interactions of the UVRABC Endonuclease in Vivo and in Vitro with DMA Damage Produced by Antineoplastic Anthracyclines1 Barry M. Kacinski2 and W. Dean Rupp3 Departments of Therapeutic Radiology [B. M. K., W. D. R.] and Molecular Biophysics and Biochemistry [W. D. R.], Yale University School of Medicine, New Haven, Connecticut 06510 ABSTRACT clines. However, they do not bind to DNA (31, 34) and do not appear to enter cells to release free drug (31, 32, 34). The anthracycline antineoplastic agents Adriamycin and N- Therefore, Tritton ef al. (30,31 ) and others (22) have proposed trifluoroacetyl-Adriamycin-14-valerate were assayed in vivo and that anthracyclines exert their antitumor effects at the cell surface in vitro for ability to produce DMA lesions recognized by the by generating free radical species which damage membranes. It UVRABC endonuclease, a DNA repair enzyme of Escherichia has also been suggested that they may interfere with nucleic coli which recognizes large, bulky lesions in DNA. We found that, acid metabolism less directly by blocking transcription of RNA while both drugs produce DNA lesions, only the lesions produced from DNA (6, 7, 27). Since data on the biochemical nature of the by Adriamycin were toxic. Hence, anthracycline antineoplastic damage to DNA induced by anthracycline exposure in mammal activity may be related to production of large, bulky lesions in ian cells are scanty (18), it is not yet possible to rule out DNA, while toxicity may correlate with toxicity measured in a anthracycline-DNA interactions in the antineoplastic activity of simple E.
    [Show full text]
  • Cho Endonuclease Functions During DNA Interstrand Cross-Link Repair in Escherichia Coli
    crossmark Cho Endonuclease Functions during DNA Interstrand Cross-Link Repair in Escherichia coli Anthonige Vidya Perera, James Brian Mendenhall, Charmain Tan Courcelle, Justin Courcelle Department of Biology, Portland State University, Portland, Oregon, USA ABSTRACT DNA interstrand cross-links are complex lesions that covalently link both strands of the duplex DNA. Lesion removal is pro- posed to be initiated via the UvrABC nucleotide excision repair complex; however, less is known about the subsequent steps of Downloaded from this complex repair pathway. In this study, we characterized the contribution of nucleotide excision repair mutants to survival in the presence of psoralen-induced damage. Unexpectedly, we observed that the nucleotide excision repair mutants exhibit differ- ential sensitivity to psoralen-induced damage, with uvrC mutants being less sensitive than either uvrA or uvrB. We show that Cho, an alternative endonuclease, acts with UvrAB and is responsible for the reduced hypersensitivity of uvrC mutants. We find that Cho’s contribution to survival correlates with the presence of DNA interstrand cross-links, rather than monoadducts, and operates at a step after, or independently from, the initial incision during the global repair of psoralen DNA adducts from the genome. http://jb.asm.org/ IMPORTANCE DNA interstrand cross-links are complex lesions that covalently bind to both strands of the duplex DNA and whose mechanism of repair remains poorly understood. In this study, we show that Cho, an alternative endonuclease, acts with UvrAB and partici- pates in the repair of DNA interstrand cross-links formed in the presence of photoactivated psoralens. Cho’s contribution to survival correlates with the presence of DNA interstrand cross-links and operates at a step after, or independently from, the ini- tial incision during the repair process.
    [Show full text]
  • DNA Repair and Recombination
    DNA Repair and Recombination Evangelia Morou-Bermudez, D.D.S., M.S., Ph.D Mutations and their biomedical importance Mutation is defined as a permanent change in the DNA sequence. Mutations can occur spontaneously from faulty replication or repair of DNA, or can be induced by external agents, such as radiation, chemicals and viruses. Mutations in germ cells are transmitted to the offspring (“vertical” transmission of hereditary disease). Mutations in somatic cells lead to an abnormal somatic cell clone (“horizontal” transmission, within the same organism). Most dangerous somatic mutations are those that cause cells to grow at increased rates, leading to neoplastic diseases. Homozygosity is the condition in which an abnormal gene is present in both copies in a diploid cell, without its normal counterpart. Heterozygosity is the condition in which the abnormal gene is present in one copy together with its normal counterpart in a diploid cell. Heterozygotes for a normal and a defective gene usually produce both the normal and the defective proteins. Dominant inheritance pattern: the heterozygous cariers of an abnormal gene are affected clinically. Recessive inheritance pattern: the heterozygous cariers of an abnormal gene are not affected clinically. Types of DNA damage and their causes. Tautomeric shifts in the bases. eg: Thymine, in normal keto form pairs with adenine. In the enol form it pairs with guanine. Pyrimidine dimers. Mostly caused by UV radiation. Types of DNA damage and their causes (cont) AP sites (apurinic or apyrimidinic). Caused by spontaneous hydrolysis of N- glycosidic bond between a purine or pyrimidine with 2-deoxyribose. Base analogues Eg.
    [Show full text]
  • Cho Endonuclease Functions During DNA Interstrand Crosslink Repair in Escherichia Coli
    Portland State University PDXScholar Biology Faculty Publications and Presentations Biology 11-2016 Cho Endonuclease Functions During DNA Interstrand Crosslink Repair in Escherichia coli Anthonige Vidya Perera Portland State University, [email protected] James Brian Mendenhall Portland State University Charmain T. Courcelle Portland State University Justin Courcelle Portland State University, [email protected] Follow this and additional works at: https://pdxscholar.library.pdx.edu/bio_fac Part of the Biology Commons Let us know how access to this document benefits ou.y Citation Details Perera, A. V., Mendenhall, J. B., Courcelle, C. T., & Courcelle, J. (2016). Cho Endonuclease Functions during DNA Interstrand Cross-Link Repair in Escherichia coli. Journal of Bacteriology, 198(22), 3099-3108. This Post-Print is brought to you for free and open access. It has been accepted for inclusion in Biology Faculty Publications and Presentations by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: [email protected]. JB Accepted Manuscript Posted Online 29 August 2016 J. Bacteriol. doi:10.1128/JB.00509-16 1 TITLE: Cho endonuclease functions during DNA interstrand crosslink repair in 2 Escherichia coli 3 4 RUNNING TITLE: Cho functions during DNA interstrand crosslink repair 5 Anthonige Vidya Perera, James Brian Mendenhall, Charmain Tan Courcelle, and Justin Downloaded from 6 Courcelle* 7 Department of Biology, Portland State University, Portland, OR 97201 8 *email: [email protected] 9 key words: Cho, Nucleotide excision repair, DNA interstrand crosslink, psoralen 10 http://jb.asm.org/ 11 ABSTRACT 12 DNA interstrand crosslinks are complex lesions that covalently link both strands 13 of the duplex DNA.
    [Show full text]
  • Enzymatic Properties of Purified Escherichia Coliuvrabc Proteins
    Proc. Nati. Acad. Sci. USA Vol. 80, pp. 6157-6161, October 1983 Biochemistry Enzymatic properties of purified Escherichia coli uvrABC proteins (DNA repair/UV endonuclease/uvrA/uvrB/uvrC) ANTHONY T. YEUNG*, WILLIAM B. MATTES, EUK Y. OH, AND LAWRENCE GROSSMANt Department of Biochemistry, The Johns Hopkdns University School of Hygiene and Public Health, Baltimore, MD 21205 Communicated by Hamilton 0. Smith, June 22, 1983 ABSTRACT The cloned uwrA and uvrB genes of Escherichia METHODS coli K-12 were amplified by linkage to the PL promoter of plasmid uvrC Proteins. pKC30. The uvrC gene was amplified in the high-copy-number Purification of the uvrA, uvrB, and Having plasmid pRLM 24. The three gene products (purified in each case cloned the uvrA, uvrC (12), and uvrB (unpublished data) genes to greater than 95% purity) and ATP are required to effectively from the chromosome of E. coli strain AB1157 we made use of incise UV-damaged DNAs. The uvrABC proteins bind tightly to the strong PL promoter of phage A to amplify the uvrA protein damaged sites in DNA, requiring the initial atachment of the uvrA to 7% of the total protein of E. coli (13) and the uvrB protein protein in the presence of ATP before productive binding of the in a similar manner to 5% of total E. coli protein (unpublished uvrB and uvrC proteins. Using a cloned tandem double insert of data). The uvrC protein was amplified about 300-fold by in- the lac p-o region as a damaged DNA substrate for the uvrABC creasing the uvrC gene dosage through its cloning in high-opy- complex and analyzing the incision both 5' and 3' to each pyrim- number plasmid pRLM24 (source: Roger L.
    [Show full text]
  • Identification of Putative Drug Targets and Vaccine Candidates For
    nalytic A al & B y i tr o s c i h e m m e Biochemistry & i h s c t Hema, et al., Biochem Anal Biochem 2015, 4:2 r o i y B DOI: 10.4172/2161-1009.1000175 ISSN: 2161-1009 Analytical Biochemistry Research Article Open Access Identification of Putative Drug Targets and Vaccine Candidates for Pathogens Causing Atherosclerosis Kanipakam Hema1, Vani Priyadarshini I1, Dibyabhaba Pradhan1, Manne Munikumar1, Swargam Sandeep1, Natarajan Pradeep1, Suchitra MM2 and Amineni Umamaheswari1* 1SVIMS Bioinformatics Centre, Department of Bioinformatics, SVIMS University, Tirupati-517507, AP, India 2Department of Biochemistry, SVIMS University, Tirupati-517507, AP, India *Corresponding author: Umamaheswari A, Associate Professor and Coordinator of BIF SVIMS Bioinformatics Centre Department of Bioinformatics SVIMS University, Tirupati – 517507, AP, India, Tel: 0877-2287727; E-mail: [email protected] Rec date: Apr 07, 2015; Acc date: Apr 15, 2015; Pub date: Apr 17, 2015 Copyright: © 2015 Hema K, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Atherosclerosis is a chronic inflammatory artery disease, responsible for both cardiovascular (heart) and cerebrovascular (brain) stroke with high morbidity and mortality worldwide. Infectious pathogens such as Chlamydophila pneumoniae, Porphyromonas gingivalis and Helicobacter pylori are shown to be associated with the disease in recent epidemiological studies. Therefore, identification of common drug targets and vaccine candidates against these three pathogens would be vital towards therapy and management of atherosclerosis. Chlamydophila pneumonia was selected as a reference organism due to its predominant role in atherosclerosis.
    [Show full text]
  • Removal of Misincorporated Ribonucleotides from Prokaryotic Genomes: an Unexpected Role for Nucleotide Excision Repair
    Removal of Misincorporated Ribonucleotides from Prokaryotic Genomes: An Unexpected Role for Nucleotide Excision Repair Alexandra Vaisman1, John P. McDonald1, Donald Huston1, Wojciech Kuban1¤, Lili Liu2, Bennett Van Houten2, Roger Woodgate1* 1 Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America, 2 Department of Pharmacology and Chemical Biology, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, United States of America Abstract Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V) has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A) reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER). We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in DuvrA, uvrB5 and DuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER) in vivo.
    [Show full text]
  • Chapter 30: DNA Replication, Repair, and Recombination
    Chapter 30: DNA Replication, Repair, and Recombination 1. DNA Replication: An overview 2. Enzymes of Replication 3. Prokaryotic Replication 4. Eukaryotic Replication 5. Repair of DNA 6. Recombination and Mobile Genetic Elements 7. DNA Methylation and Trinucleotide Repeat Expansion DNA Replication • DNA double strand -> template for duplication, Replication • Chemically similar to transcription • As complex as translation but enzymes in only few copies/cell • Extremely accurate: 10-10 mistakes/base • Extremely regulated: only once per cell division Action of DNA polymerase • Template • dNTPs • 5’ -> 3’ direction • Semi-conservative Replication of DNA Unwinding of dsDNA: - Rate in E. coli: 1000nt/sec - 100rev/sec (10bp/turn) Negative supercoils by: DNA Gyrase type II topoisomerase, ATP E. coli theta replication • autoradiogramm • branch point called “replication fork” • unidirectional / bidirectional • prokaryotes and bacteriophages have only one origin of replication Unidirectional vs. bidirectional θ replication [3H]tymidine pulse-labelling Semidiscontinuous DNA replication Okazaki fragments: Discontinous ! 1000-2000nt in prokaryotes 100-200nt in eukaryotes Joined by DNA ligase Replication eye in Drosophila melanogaster DNA Priming of DNA synthesis by short RNA segments E. coli: RNA Polymerase Primase, rifampicin sensitive Removal of RNA primers 2. Enzymes of replication DNA Replication requires (in order of appearance): 1. DNA Topoisomerase 2. Helicases 3. ssDNA binding proteins 4. RNA primer synthesis 5. DNA polymerase 6. Enzyme to remove RNA primers 7. Link Okazaki fragments E. coli DNA polymerase I in complex with a dsDNA Arthur Kornberg, 1957 DNA Polymerase I 5’->3’ synthesis Processive, 20nt Recognizes dNTP based on base pairing Right hand sructure Editing activity: 3’->5’ exonuclease 5’->3’ exonuclease (proofreading) Fidelity 10-7 Klenow fragment Lacks 5’->3’ exo, lacks N- term.
    [Show full text]