DOCSLIB.ORG

Review Type II Restriction Endonucleases

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Review Type II Restriction Endonucleases CMLS, Cell. Mol. Life Sci. 62 (2005) 685–707 1420-682X/05/060685-23 DOI 10.1007/s00018-004-4513-1 CMLS Cellular and Molecular Life Sciences © Birkhäuser Verlag, Basel, 2005 Review Type II restriction endonucleases: structure and mechanism A. Pingouda,*, M. Fuxreiterb, V.Pingouda and W.Wendea a Institut für Biochemie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, 35392 Giessen (Germany), Fax: +49 641 9935409, e-mail: [email protected] b Institute of Enzymology, Biological Research Centre, Hungarian Academy of Sciences, Karolina ut 29, 1113 Budapest (Hungary) Received 15 November 2004; accepted 9 December 2004 Abstract. Type II restriction endonucleases are compo- few restriction endonucleases discovered thus far do not nents of restriction modification systems that protect belong to the PD…D/ExK family of enzymes, but rather bacteria and archaea against invading foreign DNA. Most have active sites typical of other endonuclease families. are homodimeric or tetrameric enzymes that cleave DNA The present review deals with new developments in the at defined sites of 4–8 bp in length and require Mg2+ ions field of Type II restriction endonucleases. One of the for catalysis. They differ in the details of the recognition more interesting aspects is the increasing awareness of process and the mode of cleavage, indicators that these the diversity of Type II restriction enzymes. Nevertheless, enzymes are more diverse than originally thought. Still, structural studies summarized herein deal with the more most of them have a similar structural core and seem to common subtypes. A major emphasis of this review will share a common mechanism of DNA cleavage, suggest- be on target site location and the mechanism of catalysis, ing that they evolved from a common ancestor. Only a two problems currently being addressed in the literature. Key words. Protein-nucleic acid interaction; facilitated diffusion; DNA recognition; DNA cleavage; mechanism of phosphodiester bond hydrolysis; evolution; protein engineering. Introduction among bacteria [4] and generation of genetic variation [5, 6]. Restriction endonucleases of Chlorella viruses may Restriction endonucleases are components of restriction have a nutritive function by helping degrade host DNA or modification (RM) systems that occur ubiquitously preventing infection of a cell by another virus [3]. Certain among bacteria, archaea [1, 2] and in viruses of certain types of RM systems can also be considered as selfish unicellular algae [3]. Their main function is to defend DNA elements [7, 8]. In general, bacteria and archaea their host against foreign DNA. This is achieved by cleav- harbour numerous RM systems. For example, in Heli- ing incoming DNA that is recognized as foreign by cobacter pylori more than 20 putative RM systems, the absence of a characteristic modification (N4 or C5 comprising greater than 4% of the total genome, havebeen methylation at cytosine or N6 methylation at adenine) at identified in two completely sequenced H. pylori strains defined sites within the recognition sequence. The host [9]. Several types of restriction endonucleases exist that DNA is resistant to cleavage as these sites are modified. differ in subunit composition and cofactor requirement. Additional functions have been attributed to restriction Commonly, four types are distinguished [10]. enzymes, including maintenance of species identity Type I restriction enzymes consist of three different subunits, HsdM, HsdR and HsdS, that are responsible for * Corresponding author. modification, restriction and sequence recognition, 686 A. Pingoud et al. Type II restriction endonucleases respectively. The quaternary structure of the active Type I DNA within this sequence in both strands, producing restriction enzyme is HsdM2HsdR2HsdS. Type I enzymes 3¢-hydroxyls and 5¢-phosphate ends. Some recognize require ATP, Mg2+and AdoMet for activity. They interact discontinuous palindromes, interrupted by a segment of in general with two asymmetrical bi-partite recognition specified length but unspecified sequence. The DNA sites, translocate the DNA in an ATP-hydrolysis depen- fragments produced have ‘blunt’ or ‘sticky’ ends with dent manner and cut the DNA distal to the recognition 3¢- or 5¢-overhangs of up to 5 nucleotides (there is a sites, approximately half-way between two sites. Typical single known example of an enzyme producing a 7-nu- examples are EcoKI, EcoAI, EcoR124I and StySBLI, cleotide 3¢-overhang: TspRI (CASTGNN/)). Most of the which represent Type IA, IB, IC and ID subtypes, respec- restriction enzymes used for recombinant DNA work tively [11–14]. [21–23] belong to this subtype, which is called Type IIP Type III restriction enzymes consist of two subunits only, (P for –palindromic) according to the accepted nomencla- Mod (responsible for DNA recognition and modification) ture [10]. Many Type II restriction endonucleases have and Res (responsible for DNA cleavage). Active nucleases properties different from the Type IIP enzymes, for which 2+ have a Mod2Res2 stoichiometry, require ATP and Mg for EcoRI (recognition sequence G/AATTC) and EcoRV activity and are stimulated by AdoMet. They interact with (GAT/ATC) are the best-known and best-studied repre- two head-to-head arranged asymmetrical recognition sentatives. The current nomenclature tries to group the sites, translocate the DNA in an ATP-hydrolysis depen- Type II restriction enzymes according to properties that dent manner and cut the DNA close to one recognition are unique to the respective subtype. However, as will be site. Typical examples are EcoP1I and EcoP15I [12–14]. seen, overlap cannot be avoided. This is the consequence Type IV restriction enzymes recognize and cleave methy- of the great diversity among Type II restriction endonu- lated DNA. As such they are not part of an RM system. cleases. The best-studied representative is McrBC, which consists Type IIA enzymes recognize asymmetric sequences. An of two different subunits, McrB and McrC, responsible interesting member of this subtype is Bpu10I [CCT- for DNA recognition and cleavage, respectively. McrBC NAGC(-5/-2)], a dimer of non-identical subunits, each of recognizes DNA with at least two RC sequences at a vari- which is responsible for cleavage of one strand of the able distance, containing methylated or hydroxymethy- DNA: 5¢-CC/TNAGC-3¢ and 5¢-GC/TNAGG-3¢ [24]. lated cytosine in one or both strands. For DNA cleavage These enzymes are ideal precursors for the generation of GTP and Mg2+ are required; cleavage occurs close to one nicking enzymes. of the two RmC sites [12–14]. Type IIB enzymes cleave DNA at both sides of the This review will deal with Type II restriction endonu- recognition sequence, an example being BplI cleases with a particular focus on the structure and [(8/13)GAGNNNNNCTC(13/8)]. BplI cleaves the top mechanism of these enzymes. For previous reviews see strand 8 nucleotides before and 13 nucleotides after the references [15–20]. recognition sequence, while the bottom strand is cleaved 13 nucleotides before and 8 nucleotides after the recogni- tion sequence [25]. Diversity of Type II restriction endonucleases Type IIC enzymes have both cleavage and modification domains within one polypeptide. One of the first discov- As of 29 October 2004, REBASE (http://rebase.neb.com/ ered was BcgI [(10/12)CGANNNNNNTGC(12/10)], rebase/rebase.html) lists 3707 restriction enzymes: 59 which has a very unusual functional organization: it has Type I, 3635 Type II, 10 Type III and 3 Type IV. The an A2B quaternary structure [26] with both endonuclease predominance of Type II enzymes certainly is biased by and methyltransferase domains in the A subunit and the their usefulness for recombinant DNA work. The analysis target recognition domain located in the B subunit [27]. of published genome sequences suggests a somewhat BcgI illustrates the problem of the nomenclature of Type more even distribution among putative RM systems: II restriction endonucleases, as it is also a Type IIB approximately 29% Type I, 45% Type II, 8% Type III and enzyme. 18% Type IV [R. Roberts, personal comm.]. Type IIE enzymes need to interact with two copies of Type II restriction endonucleases differ from the Type I, their recognition sequence for efficient cleavage, one III and IV enzymes by a more simplified subunit organi- copy being the target for cleavage, the other serving as an zation. They are usually homodimeric or homotetrameric allosteric effector [28–30]. The best-studied examples enzymes that cleave DNA within or close to their recog- with respect to structure and function are EcoRII nition site and do not require ATP or GTP. With only one (/CCWGG) [31-36] and NaeI (GCC/CGG) [37-41]. It is exception known to date (see below), they require Mg2+ interesting to note that the removal of the effector domain as cofactor. of EcoRII converts this Type IIE enzyme into a very Orthodox Type II enzymes are homodimers that recognizes active Type IIP enzyme [34]. Sau3AI (/GATC), in the palindromic sequences of 4–8 bp in length, and cleave absence of DNA a monomer with two similar domains, CMLS, Cell. Mol. Life Sci. Vol. 62, 2005 Review Article 687 dimerizes in the presence of DNA and then functions as a which is composed of two different subunits. The func- Type IIE enzyme, with a catalytic site and an allosteric tional restriction endonuclease presumably is a a2b2 effector site [42]. tetramer [68]. Several of these enzymes have been used to Type IIF enzymes are typically homotetrameric restric- generate nicking enzymes, viz. BbvCI, BsaI, BsmAI, tion endonucleases that also interact with two copies of BsmBI and BsrDI [69]. their recognition site, but cleave both of them in a more Some Type II restriction enzymes only nick DNA. DNA or less concerted manner [28, 30, 43, 44]. Well-studied nicking endonucleases were found in Bacillus stearother- examples are Cfr10I (R/CCGGY) [45-47], NgoMIV mophilus, for example Nt.BstNBI and Nt.BstSEI (G/CCGGC) [47, 48] and SfiI (GGCCNNNN/NGGCC) (GAGTCN4/) [70, 71], and in Chlorella viruses, for exam- [49, 50].
Load more

Recommended publications
  • Regulation of ATP Levels in Escherichia Coli Using CRISPR
    Tao et al. Microb Cell Fact (2018) 17:147 https://doi.org/10.1186/s12934-018-0995-7 Microbial Cell Factories RESEARCH Open Access Regulation of ATP levels in Escherichia coli using CRISPR interference for enhanced pinocembrin production Sha Tao, Ying Qian, Xin Wang, Weijia Cao, Weichao Ma, Kequan Chen* and Pingkai Ouyang Abstract Background: Microbial biosynthesis of natural products holds promise for preclinical studies and treating diseases. For instance, pinocembrin is a natural favonoid with important pharmacologic characteristics and is widely used in preclinical studies. However, high yield of natural products production is often limited by the intracellular cofactor level, including adenosine triphosphate (ATP). To address this challenge, tailored modifcation of ATP concentration in Escherichia coli was applied in efcient pinocembrin production. Results: In the present study, a clustered regularly interspaced short palindromic repeats (CRISPR) interference sys- tem was performed for screening several ATP-related candidate genes, where metK and proB showed its potential to improve ATP level and increased pinocembrin production. Subsequently, the repression efciency of metK and proB were optimized to achieve the appropriate levels of ATP and enhancing the pinocembrin production, which allowed the pinocembrin titer increased to 102.02 mg/L. Coupled with the malonyl-CoA engineering and optimization of culture and induction condition, a fnal pinocembrin titer of 165.31 mg/L was achieved, which is 10.2-fold higher than control strains. Conclusions: Our results introduce a strategy to approach the efcient biosynthesis of pinocembrin via ATP level strengthen using CRISPR interference. Furthermore coupled with the malonyl-CoA engineering and induction condi- tion have been optimized for pinocembrin production.
    [Show full text]
  • Restriction-Modification Gene Complexes As Selfish Gene Entities: Roles of a Regulatory System in Their Establishment, Maintenan
    Proc. Natl. Acad. Sci. USA Vol. 95, pp. 6442–6447, May 1998 Microbiology Restriction-modification gene complexes as selfish gene entities: Roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion (programmed cell deathyepigeneticsyintragenomic conflictsyphage exclusionyplasmid) Y. NAKAYAMA AND I. KOBAYASHI* Department of Molecular Biology, Institute of Medical Science, University of Tokyo, Shirokanedai, Tokyo 108-8639, Japan Communicated by Allan Campbell, Stanford University, Stanford, CA, March 6, 1998 (received for review May 31, 1997) ABSTRACT We have reported some type II restriction- cell has lost its RM gene complex, its descendants will contain modification (RM) gene complexes on plasmids resist displace- fewer and fewer molecules of the modification enzyme. Eventu- ment by an incompatible plasmid through postsegregational host ally, their capacity to modify the many sites needed to protect the killing. Such selfish behavior may have contributed to the spread newly replicated chromosomes from the remaining pool of restric- and maintenance of RM systems. Here we analyze the role of tion enzyme may become inadequate. Chromosomal DNA will regulatory genes (C), often found linked to RM gene complexes, then be cleaved at the unmodified sites, and the cells will be killed in their interaction with the host and the other RM gene com- (refs. 2–4; N. Handa, A. Ichige, K. Kusano, and I.K., unpublished plexes. We identified the C gene of EcoRV as a positive regulator results). This result is similar to the postsegregational host killing of restriction. A C mutation eliminated postsegregational killing mechanisms for maintenance of several plasmids (5). These RM by EcoRV.
    [Show full text]
  • Gibson Assembly Cloning Guide, Second Edition
    Gibson Assembly® CLONING GUIDE 2ND EDITION RESTRICTION DIGESTFREE, SEAMLESS CLONING Applications, tools, and protocols for the Gibson Assembly® method: • Single Insert • Multiple Inserts • Site-Directed Mutagenesis #DNAMYWAY sgidna.com/gibson-assembly Foreword Contents Foreword The Gibson Assembly method has been an integral part of our work at Synthetic Genomics, Inc. and the J. Craig Venter Institute (JCVI) for nearly a decade, enabling us to synthesize a complete bacterial genome in 2008, create the first synthetic cell in 2010, and generate a minimal bacterial genome in 2016. These studies form the framework for basic research in understanding the fundamental principles of cellular function and the precise function of essential genes. Additionally, synthetic cells can potentially be harnessed for commercial applications which could offer great benefits to society through the renewable and sustainable production of therapeutics, biofuels, and biobased textiles. In 2004, JCVI had embarked on a quest to synthesize genome-sized DNA and needed to develop the tools to make this possible. When I first learned that JCVI was attempting to create a synthetic cell, I truly understood the significance and reached out to Hamilton (Ham) Smith, who leads the Synthetic Biology Group at JCVI. I joined Ham’s team as a postdoctoral fellow and the development of Gibson Assembly began as I started investigating methods that would allow overlapping DNA fragments to be assembled toward the goal of generating genome- sized DNA. Over time, we had multiple methods in place for assembling DNA molecules by in vitro recombination, including the method that would later come to be known as Gibson Assembly.
    [Show full text]
  • Supplementary Table S1. Table 1. List of Bacterial Strains Used in This Study Suppl
    Supplementary Material Supplementary Tables: Supplementary Table S1. Table 1. List of bacterial strains used in this study Supplementary Table S2. List of plasmids used in this study Supplementary Table 3. List of primers used for mutagenesis of P. intermedia Supplementary Table 4. List of primers used for qRT-PCR analysis in P. intermedia Supplementary Table 5. List of the most highly upregulated genes in P. intermedia OxyR mutant Supplementary Table 6. List of the most highly downregulated genes in P. intermedia OxyR mutant Supplementary Table 7. List of the most highly upregulated genes in P. intermedia grown in iron-deplete conditions Supplementary Table 8. List of the most highly downregulated genes in P. intermedia grown in iron-deplete conditions Supplementary Figures: Supplementary Figure 1. Comparison of the genomic loci encoding OxyR in Prevotella species. Supplementary Figure 2. Distribution of SOD and glutathione peroxidase genes within the genus Prevotella. Supplementary Table S1. Bacterial strains Strain Description Source or reference P. intermedia V3147 Wild type OMA14 isolated from the (1) periodontal pocket of a Japanese patient with periodontitis V3203 OMA14 PIOMA14_I_0073(oxyR)::ermF This study E. coli XL-1 Blue Host strain for cloning Stratagene S17-1 RP-4-2-Tc::Mu aph::Tn7 recA, Smr (2) 1 Supplementary Table S2. Plasmids Plasmid Relevant property Source or reference pUC118 Takara pBSSK pNDR-Dual Clonetech pTCB Apr Tcr, E. coli-Bacteroides shuttle vector (3) plasmid pKD954 Contains the Porpyromonas gulae catalase (4)
    [Show full text]
  • Datasheet for Ecorv-HF™ (R3195; Lot 0051211)
    Source: An E. coli strain that carries the cloned Diluent Compatibility: Diluent Buffer B Endonuclease Activity: Incubation of a 50 µl and modified (D19A, E27A) EcoRV gene from the 300 mM NaCl, 10 mM Tris-HCl, 0.1 mM EDTA, reaction containing 30 units of enzyme with 1 µg ™ plasmid J62 pLG74 (L.I. Glatman) 1 mM dithiothreitol, 500 µg/ml BSA and 50% glyc- of φX174 RF I DNA for 4 hours at 37°C resulted EcoRV-HF erol (pH 7.4 @ 25°C) in < 30% conversion to RFII as determined by Supplied in: 200 mM NaCl, 10 mM Tris-HCl agarose gel electrophoresis. 1-800-632-7799 (ph 7.4), 0.1 mM EDTA, 1 mM DTT, 200 µg/ml Quality Controls [email protected] BSA and 50% glycerol. Ligation: After 10-fold overdigestion with Blue/White Screening Assay: An appropriate www.neb.com α R3195S 005121114111 EcoRV- HF, > 95% of the DNA fragments can vector is digested at a unique site within the lacZ Reagents Supplied with Enzyme: be ligated with T4 DNA Ligase (at a 5´ termini gene with a 10-fold excess of enzyme. The vector 10X NEBuffer 4. concentration of 1–2 µM) at 16°C. Of these ligated DNA is then ligated, transformed, and plated onto R3195S fragments, > 95% can be recut with EcoRV-HF. Xgal/IPTG/Amp plates. Successful expression Reaction Conditions: 1X NEBuffer 4. of β-galactosidase is a function of how intact its 4,000 units 20,000 U/ml Lot: 0051211 Incubate at 37°C. 16-Hour Incubation: A 50 µl reaction containing gene remains after cloning, an intact gene gives 1 µg of DNA and 120 units of EcoRV-HF incubated rise to a blue colony, removal of even a single RECOMBINANT Store at –20°C Exp: 11/14 1X NEBuffer 4: for 16 hours at 37°C resulted in a DNA pattern free base gives rise to a white colony.
    [Show full text]
  • CRISPR Foki Dead Cas9 System: Principles and Applications in Genome Engineering
    cells Review CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering Maryam Saifaldeen y, Dana E. Al-Ansari y , Dindial Ramotar * and Mustapha Aouida * College of Health and Life Sciences, Division of Biological and Biomedical Sciences, Hamad Bin Khalifa University, Education City, Qatar Foundation, Doha P.O.Box 34110, Qatar; [email protected] (M.S.); [email protected] (D.E.A.-A.) * Correspondence: [email protected] (D.R.); [email protected] (M.A.) These authors contributed equally to this work. y Received: 27 September 2020; Accepted: 19 November 2020; Published: 21 November 2020 Abstract: The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR–FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms.
    [Show full text]
  • Psp73 Vector Technical Bulletin #TB041
    tb041.0906.qxp 9/25/2006 10:44 AM Page a Technical Bulletin pSP73 Vector INSTRUCTIONS FOR USE OF PRODUCT P2221. PRINTED IN USA. Revised 9/06 Part# TB041 AF9TB041 0906TB041 tb041.0906.qxp 9/25/2006 10:46 AM Page 1 pSP73 Vector All technical literature is available on the Internet at: www.promega.com/tbs/ Please visit the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on use of this system. E-mail: [email protected] I. Description..........................................................................................................1 II. Product Components and Storage Conditions ............................................1 III. pSP73 Vector Multiple Cloning Region and Circle Map..........................2 IV. pSP73 Vector Restriction Sites........................................................................4 V. Related Products ................................................................................................6 VI. Reference .............................................................................................................7 I. Description The pSP73 Vector (1) offers a wide range of restriction sites, providing greater versatility in cloning and transcription of RNA in vitro. The pSP73 Vector contains the SP6 and T7 RNA polymerase promoters and a unique multiple cloning region, which includes restriction sites for BglII, EcoRV, ClaI, EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, AccI, SalI, PstI, SphI, HindIII, PvuII and XhoI. The sequences of Promega vectors are available online at www.promega.com/vectors/ and are also available from the GenBank® database. II. Product Components and Storage Conditions Product Size Cat.# pSP73 Vector 20µg P2221 Storage Conditions: Store the pSP73 Vector at –20°C. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA.
    [Show full text]
  • Bpuami: a Novel Saci Neoschizomer from Bacillus Pumilus Discovered in an Isolate from Amazon Basin, Recognizing 5'-Gag↓Ctc-3'
    Brazilian Journal of Microbiology (2006) 37:96-100 ISSN 1517-8382 BPUAMI: A NOVEL SACI NEOSCHIZOMER FROM BACILLUS PUMILUS DISCOVERED IN AN ISOLATE FROM AMAZON BASIN, RECOGNIZING 5'-GAG↓CTC-3' Jocelei M. Chies1,2,*; Ana C. de O. Dias1; Hélio M. M. Maia3; Spartaco Astolfi-Filho2,3 1Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, RS, Brasil; 2Curso de Pós-Graduação em Biologia Molecular, Universidade de Brasília, Brasília, DF, Brasil; 3Centro de Apoio Multidisciplinar, Universidade Federal do Amazonas, Manaus, AM, Brasil Submitted: June 27, 2005; Returned to authors for corrections: November 16, 2005; Approved: January 12, 2006 ABSTRACT A strain of Bacillus pumilus was isolated and identified from water samples collected from a small affluent of the Amazon River. Type II restriction endonuclease activity was detected in these bacteria. The enzyme was purified and the molecular weight of the native protein estimated by gel filtration and SDS-PAGE. The optimum pH, temperature and salt requirements were determined. Quality control assays showed the complete absence of “nonspecific nucleases.” Restriction cleavage analysis and DNA sequencing of restriction fragments allowed the unequivocal demonstration of 5´GAG↓CTC3´ as the recognition sequence. This enzyme was named BpuAmI and is apparently a neoschizomer of the prototype restriction endonuclease SacI. This is the first report of an isoschizomer and/or neoschizomer of the prototype SacI identified in the genus Bacillus. Key words: type II restriction endonuclease, BpuAmI, SacI, neoschizomers, Bacillus pumilus INTRODUCTION thousands of taxonomically diverse bacteria for enzymes with new characteristics. However, to our knowledge, there has Restriction endonucleases are enzymes which recognize not been any report to date of an isoschizomer and/or short DNA sequences and cleave DNA in both strands.
    [Show full text]
  • The Application of Transcription Activator-Like Effector Nucleases for Genome Editing in C
    Methods 68 (2014) 389–396 Contents lists available at ScienceDirect Methods journal homepage: www.elsevier.com/locate/ymeth The application of transcription activator-like effector nucleases for genome editing in C. elegans ⇑ ⇑ Peishan Yi a,b, Wei Li c, , Guangshuo Ou d, a National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China b University of Chinese Academy of Sciences, Beijing 100049, China c School of Medicine, Tsinghua University, Beijing 100084, China d Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China article info abstract Article history: The nematode Caenorhabditis elegans has been a powerful model system for biomedical research in the Received 31 December 2013 past decades, however, the efficient genetic tools are still demanding for gene knockout, knock-in or con- Revised 14 April 2014 ditional gene mutations. Transcription activator-like effector nucleases (TALENs) that comprise a Accepted 17 April 2014 sequence-specific DNA-binding domain fused to a FokI nuclease domain facilitate the targeted genome Available online 26 April 2014 editing in various cell types or organisms. Here we summarize the recent progresses and protocols using TALENs in C. elegans that generate gene mutations and knock-ins in the germ line and the conditional Keywords: gene knockout in somatic tissues. Caenorhabditis elegans Ó 2014 Elsevier Inc. All rights reserved. Transcription activator-like effector nucleases (TALENs) Mutation Conditional gene knockout 1. Introduction length at the site of the break, whereas HDR can generate precisely specified changes at the target site when a homologous DNA The newly-developed transcription activator-like effector template is provided.
    [Show full text]
  • Method for Cloning and Producing the Bglii Restriction Endonuclease
    Europäisches Patentamt (19) European Patent Office Office européen des brevets (11) EP 0 619 373 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int. Cl.7: C12N 15/55, C12N 15/54, of the grant of the patent: C12N 1/21, C12P 21/00 24.05.2000 Bulletin 2000/21 (21) Application number: 94302258.2 (22) Date of filing: 29.03.1994 (54) Method for cloning and producing the BglII restriction endonuclease and modification methylase Verfahren zur Klonierung und zur Herstellung der Restriktionsendonuklease und Modifikationsmethylase BglII Procédé de clonage et de production de l'endonucléase de restriction BglII et de la méthylase correspondante (84) Designated Contracting States: (74) Representative: DE FR GB Davies, Jonathan Mark Reddie & Grose (30) Priority: 09.04.1993 US 17664 16 Theobalds Road London WC1X 8PL (GB) (43) Date of publication of application: 12.10.1994 Bulletin 1994/41 (56) References cited: EP-A- 0 248 678 (73) Proprietor: NEW ENGLAND BIOLABS, INC. • J. BACTERIOL., vol. 134, 1978 pages 338-344, Beverly Massachusetts 01915 (US) C.H. DUNCAN ET AL.; 'Biochemical and genetic properties of site-specific restriction (72) Inventors: endonucleases in Bacillus globigii' • Brooks, Joan E. • EUR. J. BIOCHEM., vol. 117, 1981 pages 395-399, Beverly, Massachusetts 01915 (US) R. IMBER AND T.A. BICKLE; 'Purification and • Heiter, Daniel F. properties of the restriction endonuclease BglII Groveland, Massachusetts 01834 (US) from Bacillus globigii' • Anton, Brian P. Boston MA 02116-1203 (US) Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted.
    [Show full text]
  • Restriction Endonucleases
    Restriction Endonucleases TECHNICAL GUIDE UPDATE 2017/18 be INSPIRED drive DISCOVERY stay GENUINE RESTRICTION ENZYMES FROM NEB Cut Smarter with Restriction Enzymes from NEB® Looking to bring CONVENIENCE to your workflow? Simplify Reaction Setup and Double Activity of DNA Modifying Enzymes in CutSmart Buffer: Digestion with CutSmart® Buffer Clone Smarter! Activity Enzyme Required Supplements Over 210 restriction enzymes are 100% active in a single buffer, in CutSmart Phosphatases: CutSmart Buffer, making it significantly easier to set up your Alkaline Phosphatase (CIP) + + + double digest reactions. Since CutSmart Buffer includes BSA, there Antarctic Phosphatase + + + Requires Zn2+ Quick CIP + + + are fewer tubes and pipetting steps to worry about. Additionally, Shrimp Alkaline Phosphatase (rSAP) + + + many DNA modifying enzymes are 100% active in CutSmart Ligases: T4 DNA Ligase + + + Requires ATP Buffer, eliminating the need for subsequent purification. E. coli DNA Ligase + + + Requires NAD T3 DNA Ligase + + + Requires ATP + PEG For more information, visit www.NEBCutSmart.com T7 DNA Ligase + + + Requires ATP + PEG Polymerases: T4 DNA Polymerase + + + DNA Polymerase I, Large (Klenow) Frag. + + + DNA Polymerase I + + + DNA Polymerase Klenow Exo– + + + Bst DNA Polymerase + + + ™ phi29 DNA Polymerase + + + Speed up Digestions with Time-Saver T7 DNA Polymerase (unmodified) + + + Qualified Restriction Enzymes Transferases/Kinases: T4 Polynucleotide Kinase + + + Requires ATP + DTT T4 PNK (3´ phosphatase minus) + + + Requires ATP + DTT > 190 of our restriction enzymes are able to digest DNA in CpG Methyltransferase (M. SssI) + + + 5–15 minutes, and can safely be used overnight with no loss of GpC Methyltransferase (M. CviPI) + Requires DTT T4 Phage β-glucosyltransferase + + + sample. For added convenience and flexibility, most of these are Nucleases, other: supplied with CutSmart Buffer.
    [Show full text]
  • Site-Specific DNA Targeting: Genome Engineering Tools Andrew Kuznetsov Freiburg I.Br
    Site-specific DNA targeting: genome engineering tools Andrew Kuznetsov Freiburg i.Br. 17.09.2015 The Department of Biophysics at the University of Sevastopol Content • Nucleases type II, IIS, meganucleases (EcoRV, FokI, I-SceI), mechanism of DNA cleavage • Artificial nucleases Waclaw Szybalski‘s idea, monopod design, bipod design • Advanced approaches Zinc fingers, TALEN, CRISPR/Cas9 • Applications regulation of transcription, epigenetic modification, site-specific recombination, multiplexing Types of restriction endonucleases I. (EC 3.1.21.3) cleave at sites remote from recognition site; require ATP and S-adenosyl-L-methionine to function; multifunctional proteins with restriction and methylase (EC 2.1.1.72) activities II. (EC 3.1.21.4) cleave within or at short specific distances from recognition site; most require magnesium; single function (restriction) III. (EC 3.1.21.5) cleave at a short distance from recognition site; require ATP (but do not hydrolyse it); S-adenosyl-L-methionine stimulates reaction but is not required; exist as part of a complex with a modification methylase (EC 2.1.1.72) IV. target modified DNA, e.g. methylated, hydroxymethylated and glucosyl- hydroxymethylated DNA V. utilize guide RNAs to target specific non-palindromic sequences found on invading DNA (e.g., the cas9-gRNA complex from CRISPRs) Features of the type II endonucleases > 3000 (600) http://rebase.neb.com Type II endonucleases recognize shot, usually palindromic, sequences of 4-8 bp Structures of restriction enzymes show a common core comprising 4
    [Show full text]