Recbcd Enzyme “Chi Recognition” Mutants Recognize Chi Recombination Hotspots in the Right DNA Context
Total Page:16
File Type:pdf, Size:1020Kb
Load more
Recommended publications
-
The Link Between Independent Acquisition of Intracellular Gamma-Endosymbionts and Concerted Evolution in Tremblaya Princeps
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Frontiers - Publisher Connector ORIGINAL RESEARCH published: 25 June 2015 doi: 10.3389/fmicb.2015.00642 The link between independent acquisition of intracellular gamma-endosymbionts and concerted evolution in Tremblaya princeps Sergio López-Madrigal 1, Amparo Latorre 1, 2, Andrés Moya 1, 2 and Rosario Gil 1* 1 Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de València, València, Spain, 2 Área de Genómica y Salud de la Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (FISABIO) – Salud Pública, València, Spain Many insect species establish mutualistic symbiosis with intracellular bacteria that complement their unbalanced diets. The betaproteobacterium “Candidatus Tremblaya” maintains an ancient symbiosis with mealybugs (Hemiptera: Pseudococcidae), which are classified in subfamilies Phenacoccinae and Pseudococcinae. Most Phenacoccinae Edited by: mealybugs have “Candidatus Tremblaya phenacola” as their unique endosymbiont, while Joerg Graf, University of Connecticut, USA most Pseudococcinae mealybugs show a nested symbiosis (a bacterial symbiont placed Reviewed by: inside another one) where every “Candidatus Tremblaya princeps” cell harbors several John M. Chaston, cells of a gammaproteobacterium. Genomic characterization of the endosymbiotic Brigham Young University, USA consortium from Planococcus citri, composed by “Ca. Tremblaya princeps” and Huan Qiu, Rutgers University, USA “Candidatus Moranella endobia,” unveiled several atypical features of the former’s *Correspondence: genome, including the concerted evolution of paralogous loci. Its comparison with the Rosario Gil, genome of “Ca. Tremblaya phenacola” PAVE, single endosymbiont of Phenacoccus Institut Cavanilles de Biodiversitat i avenae, suggests that the atypical reductive evolution of “Ca. -
Characterization of the Multidrug Resistance Protein Expressed in Cell Clones Stably Transfected with the Mouse Mdrl Cdna1
[CANCER RESEARCH 49, 2729-2734, May 15, 1989] Characterization of the Multidrug Resistance Protein Expressed in Cell Clones Stably Transfected with the Mouse mdrl cDNA1 Erwin Schurr,2 Martine Raymond,3 John C. Bell,4 and Philippe Gros5 Department of Biochemistry, Faculty of Medicine, McGill University, Montreal, Canada, H3GIY6 ABSTRACT of the nucleotide and predicted amino acid sequences of this clone indicated that the encoded polypeptide was most likely a Structural features of the multidrug resistance protein encoded by the membrane glycoprotein, highly similar or identical to P-glyco mouse mdrl gene were studied in multidrug-resistant cell clones stably protein, which contained 12 putative transmembrane domains transfected with a biologically active cDNA clone. Independently derived and a cluster of /V-linked glycosylation sites near the amino transfectant cell clones, initially selected in Adriamycin, were shown to be cross-resistant to several drugs, including actinomycin D, amsacrine, terminus. It is formed by the internal duplication of a structural mitoxantrone, VP-16, and vinblastine but remained sensitive to cis- unit which encodes three transmembrane loops and a putative platinum, 5-fluorouracil, ¡trabinocytosine, and bleomycin. In drug-resist nucleotide binding fold (7). The duplicated segment is highly ant transfectants the mdrl gene product was greatly overexpressed as a homologous to the energy coupling subunit of bacterial trans polypeptide of apparent molecular weight 160,000-170,000. This protein port proteins particularly HlyB, a protein that participates in was present in membrane enriched fractions and could be metabolically the extracellular transport of haemolysin in Escherichia coli labeled with |3H]glucosamine, confirming that the transfected mdrl gene (14). -
Restriction Endonucleases
Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic) 9 Restriction Endonucleases Derek Robinson, Paul R. Walsh, and Joseph A. Bonventre Background Information . 226 Which Restriction Enzymes Are Commercially Available? . 226 Why Are Some Enzymes More Expensive Than Others? . 227 What Can You Do to Reduce the Cost of Working with Restriction Enzymes? . 228 If You Could Select among Several Restriction Enzymes for Your Application, What Criteria Should You Consider to Make the Most Appropriate Choice? . 229 What Are the General Properties of Restriction Endonucleases? . 232 What Insight Is Provided by a Restriction Enzyme’s Quality Control Data? . 233 How Stable Are Restriction Enzymes? . 236 How Stable Are Diluted Restriction Enzymes? . 236 Simple Digests . 236 How Should You Set up a Simple Restriction Digest? . 236 Is It Wise to Modify the Suggested Reaction Conditions? . 237 Complex Restriction Digestions . 239 How Can a Substrate Affect the Restriction Digest? . 239 Should You Alter the Reaction Volume and DNA Concentration? . 241 Double Digests: Simultaneous or Sequential? . 242 225 Genomic Digests . 244 When Preparing Genomic DNA for Southern Blotting, How Can You Determine If Complete Digestion Has Been Obtained? . 244 What Are Your Options If You Must Create Additional Rare or Unique Restriction Sites? . 247 Troubleshooting . 255 What Can Cause a Simple Restriction Digest to Fail? . 255 The Volume of Enzyme in the Vial Appears Very Low. Did Leakage Occur during Shipment? . 259 The Enzyme Shipment Sat on the Shipping Dock for Two Days. -
DNA Unwinding Step-Size of E. Coli Recbcd Helicase Determined from Single Turnover Chemical Quenched-flow Kinetic Studies
B doi:10.1016/S0022-2836(02)01067-7 available online at http://www.idealibrary.com on w J. Mol. Biol. (2002) 324, 409–428 DNA Unwinding Step-size of E. coli RecBCD Helicase Determined from Single Turnover Chemical Quenched-flow Kinetic Studies Aaron L. Lucius1, Alessandro Vindigni1, Razmic Gregorian1 Janid A. Ali1, Andrew F. Taylor2, Gerald R. Smith2 and Timothy M. Lohman1* 1Department of Biochemistry The mechanism by which Escherichia coli RecBCD DNA helicase unwinds and Molecular Biophysics duplex DNA was examined in vitro using pre-steady-state chemical Washington University School quenched-flow kinetic methods. Single turnover DNA unwinding experi- of Medicine, 660 S. Euclid Ave. ments were performed by addition of ATP to RecBCD that was pre- Box 8231, St. Louis, MO 63110 bound to a series of DNA substrates containing duplex DNA regions USA ranging from 24 bp to 60 bp. In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that 2Fred Hutchinson Cancer increased with duplex length, reflecting the transient formation of Research Center, 1100 Fairview partially unwound DNA intermediates during unwinding catalyzed by Ave. North, Seattle, WA 98109 RecBCD. Quantitative analysis of five independent sets of DNA unwind- USA ing time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding “step-size”, m ¼ 3.9(^1.3) bp step21, ^ 21 with an average unwinding rate of kU ¼ 196( 77) steps s ^ 21 (mkU ¼ 790( 23) bp s ) at 25.0 8C (10 mM MgCl2, 30 mM NaCl (pH 7.0), 5% (v/v) glycerol). -
Phosphate Steering by Flap Endonuclease 1 Promotes 50-flap Specificity and Incision to Prevent Genome Instability
ARTICLE Received 18 Jan 2017 | Accepted 5 May 2017 | Published 27 Jun 2017 DOI: 10.1038/ncomms15855 OPEN Phosphate steering by Flap Endonuclease 1 promotes 50-flap specificity and incision to prevent genome instability Susan E. Tsutakawa1,*, Mark J. Thompson2,*, Andrew S. Arvai3,*, Alexander J. Neil4,*, Steven J. Shaw2, Sana I. Algasaier2, Jane C. Kim4, L. David Finger2, Emma Jardine2, Victoria J.B. Gotham2, Altaf H. Sarker5, Mai Z. Her1, Fahad Rashid6, Samir M. Hamdan6, Sergei M. Mirkin4, Jane A. Grasby2 & John A. Tainer1,7 DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 50-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 50-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 50polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via ‘phosphate steering’, basic residues energetically steer an inverted ss 50-flap through a gateway over FEN1’s active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 50-flap specificity and catalysis, preventing genomic instability. 1 Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. -
Capture of a Recombination Activating Sequence from Mammalian Cells
Gene Therapy (1999) 6, 1819–1825 1999 Stockton Press All rights reserved 0969-7128/99 $15.00 http://www.stockton-press.co.uk/gt Capture of a recombination activating sequence from mammalian cells P Olson and R Dornburg The Dorrance H Hamilton Laboratories, Center for Human Virology, Division of Infectious Diseases, Jefferson Medical College, Thomas Jefferson University, Jefferson Alumni Hall, 1020 Locust Street, Rm 329, Philadelphia, PA 19107, USA We have developed a genetic trap for identifying revealed a putative recombination signal (CCCACCC). sequences that promote homologous DNA recombination. When this heptamer or an abbreviated form (CCCACC) The trap employs a retroviral vector that normally disables were reinserted into the vector, they stimulated vector itself after one round of replication. Insertion of defined repair and other DNA rearrangements. Mutant forms of DNA sequences into the vector induced the repair of a 300 these oligomers (eg CCCAACC or CCWACWS) did not. base pair deletion, which restored its ability to replicate. Our data suggest that the recombination events occurred Tests of random sequence libraries made in the vector within 48 h after transfection. Keywords: recombination; retroviral vector; vector stability; gene conversion; gene therapy Introduction scripts are still made from the intact left LTR, but reverse transcription copies the deletion to both LTRs, disabling Recognition of cis-acting DNA signals occupies a central the daughter provirus.15–17 We found that the SIN vectors role in both site-specific and general recombination path- that could escape this programmed disablement did so 1–6 ways. Signals in site-specific pathways define the by recombinationally repairing the U3-deleted LTR. -
Thermodynamics of DNA Binding by DNA Polymerase I and Reca
Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2014 Thermodynamics of DNA Binding by DNA Polymerase I and RecA Recombinase from Deinococcus radiodurans Jaycob Dalton Warfel Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Warfel, Jaycob Dalton, "Thermodynamics of DNA Binding by DNA Polymerase I and RecA Recombinase from Deinococcus radiodurans" (2014). LSU Doctoral Dissertations. 2382. https://digitalcommons.lsu.edu/gradschool_dissertations/2382 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. THERMODYNAMICS OF DNA BINDING BY DNA POLYMERASE I AND RECA RECOMBINASE FROM DEINOCOCCUS RADIODURANS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biological Sciences by Jaycob Dalton Warfel B.S. Louisiana State University, 2006 May 2015 ACKNOWLEDGEMENTS I would like to express my utmost gratitude to the myriad of individuals who have lent their support during the time it has taken to complete this dissertation. First and foremost is due glory to God, The Father, The Son and The Holy Spirit, through whom all is accomplished. It is with extreme thankfulness for the blessings bestowed upon me, and with vast appreciation for the beauty of God’s creation that I have pursued a scientific education. -
S1 Nuclease Degrades Single-Stranded Nucleic Acids, Releasing 5'-Phosphoryl Mono- Or Oligonucleotides
Description S1 Nuclease degrades single-stranded nucleic acids, releasing 5'-phosphoryl mono- or oligonucleotides. It is five times more active on DNA than on RNA (1). S1 Nuclease also cleaves dsDNA at the single-stranded region caused by a nick, gap, mismatch or loop. PRODUCT INFORMATION S1 Nuclease exhibits 3’-phosphomonoesterase activity. S1 Nuclease The enzyme is a glycoprotein with a carbohydrate content of 18%. Pub. No. MAN0013722 Applications Rev. Date 29 November 2016 (Rev. A.00) Removal of single-stranded overhangs of DNA #_ fragments (2). S1 transcript mapping (3, 4). Lot: _ Expiry Date: _ Cleavage of hairpin loops. Creation of unidirectional deletions in DNA fragments in Store at -20 °C conjunction with Exo III (5). Source Aspergillus oryzae cells. Components #EN0321 100 U/µL S1 Nuclease 10000 U 5X Reaction Buffer 2 1 mL www.thermofisher.com For Research Use Only. Not for use in diagnostic procedures. Definition of Activity Unit CERTIFICATE OF ANALYSIS One unit of the enzyme produces 1 µg of acid soluble S1 Nuclease was tested for the absence of deoxyribonucleotides in 1 min at 37 °C. contaminating double-stranded DNA specific nuclease Enzyme activity is assayed in the following mixture: activity. 30 mM sodium-acetate (pH 4.5), 50 mM NaCl, 0.1 mM ZnCl2, 5% (v/v) glycerol, 800 µg/mL heat Quality authorized by: Jurgita Zilinskiene denatured calf thymus DNA. Storage Buffer The enzyme is supplied in: 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.1 mM ZnCl2 and 50% (v/v) glycerol. 5X Reaction Buffer 200 mM sodium acetate (pH 4.5 at 25 °C), 1.5 M NaCl and 10 mM ZnSO4. -
Recombination Hotspot ANDREW F
Proc. Natd. Acad. Sci. USA Vol. 89, pp. 5226-5230, June 1992 Biochemistry RecBCD enzyme is altered upon cutting DNA at a Chi recombination hotspot ANDREW F. TAYLOR AND GERALD R. SMITH Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, WA 98104 Communicated by Hamilton 0. Smith, March 16, 1992 ABSTRACT During its unidirectional unwinding of DNA, to ensure even numbers of exchanges in conjugal and trans- RecBCD enzyme cuts one DNA strand near a properly oriented ductional crosses (13). At each end ofthe donor fragment one Chi site, a hotspot of homologous genetic recombination in RecBCD molecule is proposed to enter and promote just one Escherichia cohl. We report here that individual DNA molecules exchange. Such a mechanism would ensure exactly two containing two properly oriented Chi sites were cut with about exchanges and, hence, viability. 40% efficiency at one or the other Chi site but not detectably By what mechanism might RecBCD promote just one at both Chi sites. Furthermore, initial incubation of RecBCD exchange near each end ofthe donor fragment? This problem with Chi-containing DNA reduced its ability both to unwind is compounded by the high density of Chi sites in E. coli DNA and to cut at Chi sites on subsequently added DNA DNA: Chi occurs, on the average, once every 5 kilobases (kb) molecules much more than did initial incubation with Chi-free (18). A conjugational donor fragment one-quarter of the DNA; the nuclease activity was less severely affected. These chromosome long would contain about 250 Chi sites. Coor- results imply that RecBCD loses its Chi-cutting activity upon dination among the multitude of potential exchanges at these cutting at a single Chi site and provide a mechanism for sites seems difficult. -
FAN1 Nuclease Activity Affects CAG Expansion and Age at Onset of Huntington's Disease
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.13.439716; this version posted April 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. McAllister, Donaldson et al FAN1 nuclease activity affects CAG expansion and age at onset of Huntington's disease Branduff McAllister, PhD1+, Jasmine Donaldson, PhD1+, Caroline S. Binda, PhD1, Sophie Powell, BSc1, Uroosa Chughtai, BSc1, Gareth Edwards, PhD1, Joseph Stone, BA1, Sergey Lobanov, PhD1, Linda Elliston, MPhil1, Laura-Nadine Schuhmacher, PhD1, Elliott Rees, PhD1, Georgina Menzies, PhD2, Marc Ciosi, PhD3, Alastair Maxwell, PhD3, Michael J. Chao, PhD4, Eun Pyo Hong, PhD4, Diane Lucente, MS4, Vanessa Wheeler, PhD4, Jong-Min Lee, PhD4,5, Marcy E. MacDonald, PhD4,5, Jeffrey D. Long, PhD6, Elizabeth H. Aylward, PhD7, G. Bernhard Landwehrmeyer, MD PhD8, Anne E. Rosser, MB BChir, PhD9,10, REGISTRY Investigators of the European Huntington’s disease network11, Jane S. Paulsen, PhD12, PREDICT-HD Investigators of the Huntington Study Group13, Nigel M. Williams, PhD1, James F. Gusella, PhD4,5, Darren G. Monckton, PhD3, Nicholas D. Allen, PhD2, Peter Holmans, PhD1, Lesley Jones, PhD1,14* & Thomas H. Massey, BM BCh, DPhil1,15* 1 Division of Psychological Medicine and Clinical Neurosciences, Cardiff University, Cardiff, CF24 4HQ, United Kingdom 2 School of Biosciences, Cardiff University, Cardiff, CF10 3AX, -
Insights from the Reanalysis of High-Throughput Chemical Genomics Data For
bioRxiv preprint doi: https://doi.org/10.1101/2020.07.16.206243; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 1 Title: Insights from the reanalysis of high-throughput chemical genomics data for 2 Escherichia coli K-12 3 Authors: Peter I-Fan Wu1, Curtis Ross1, Deborah A. Siegele2 and James C. Hu1,3 4 5 Affiliations: 6 1. Department of Biochemistry and Biophysics, Texas A&M University and Texas 7 Agrilife Research, College Station, TX 77843-2128 8 2. Department of Biology, Texas A&M University, College Station, TX 77843-3258 9 3. Deceased 10 11 Correspondence: [email protected] 12 13 14 Key words: phenotypic profiling, functional genomics, microbial genomics, biostatistics, 15 Escherichia coli, bacterial genetics 16 17 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.16.206243; this version posted July 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 2 18 ABSTRACT 19 Despite the demonstrated success of genome-wide genetic screens and chemical 20 genomics studies at predicting functions for genes of unknown function or predicting 21 new functions for well-characterized genes, their potential to provide insights into gene 22 function hasn't been fully explored. -
Structure and Function of Nucleases in DNA Repair: Shape, Grip and Blade of the DNA Scissors
Oncogene (2002) 21, 9022 – 9032 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Structure and function of nucleases in DNA repair: shape, grip and blade of the DNA scissors Tatsuya Nishino1 and Kosuke Morikawa*,1 1Department of Structural Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan DNA nucleases catalyze the cleavage of phosphodiester mismatched nucleotides. They also recognize the bonds. These enzymes play crucial roles in various DNA replication or recombination intermediates to facilitate repair processes, which involve DNA replication, base the following reaction steps through the cleavage of excision repair, nucleotide excision repair, mismatch DNA strands (Table 1). repair, and double strand break repair. In recent years, Nucleases can be regarded as molecular scissors, new nucleases involved in various DNA repair processes which cleave phosphodiester bonds between the sugars have been reported, including the Mus81 : Mms4 (Eme1) and the phosphate moieties of DNA. They contain complex, which functions during the meiotic phase and conserved minimal motifs, which usually consist of the Artemis : DNA-PK complex, which processes a V(D)J acidic and basic residues forming the active site. recombination intermediate. Defects of these nucleases These active site residues coordinate catalytically cause genetic instability or severe immunodeficiency. essential divalent cations, such as magnesium, Thus, structural biology on various nuclease actions is calcium, manganese or zinc, as a cofactor. However, essential for the elucidation of the molecular mechanism the requirements for actual cleavage, such as the types of complex DNA repair machinery. Three-dimensional and the numbers of metals, are very complicated, but structural information of nucleases is also rapidly are not common among the nucleases.