DOCSLIB.ORG

Restriction-Modification System with Methyl-Inhibited Base Excision and Abasic-Site Cleavage Activities

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Restriction-Modification System with Methyl-Inhibited Base Excision and Abasic-Site Cleavage Activities Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Fukuyo, M., T. Nakano, Y. Zhang, Y. Furuta, K. Ishikawa, M. Watanabe-Matsui, H. Yano, T. Hamakawa, H. Ide, and I. Kobayashi. “Restriction-Modification System with Methyl-Inhibited Base Excision and Abasic-Site Cleavage Activities.” Nucleic Acids Research 43, no. 5 (February 19, 2015): 2841–2852. As Published http://dx.doi.org/10.1093/nar/gkv116 Publisher Oxford University Press Version Final published version Citable link http://hdl.handle.net/1721.1/98182 Terms of Use Creative Commons Attribution Detailed Terms http://creativecommons.org/licenses/by/4.0/ Published online 19 February 2015 Nucleic Acids Research, 2015, Vol. 43, No. 5 2841–2852 doi: 10.1093/nar/gkv116 Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities Masaki Fukuyo1,2,3,4,†, Toshiaki Nakano5,†, Yingbiao Zhang1,2, Yoshikazu Furuta1,2, Ken Ishikawa1,2,6, Miki Watanabe-Matsui1,2, Hirokazu Yano1,2, Takeshi Hamakawa5, Hiroshi Ide5 and Ichizo Kobayashi1,2,6,* 1Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Tokyo 108-8639, Japan, 2Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan, 3Department of Evolutionary Studies of Biosystems, School of Advanced Sciences, The Graduate University for Advanced Studies (SOKENDAI), Hayama, Kanagawa 240-0193, Japan, 4Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan, 5Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University Higashi-Hiroshima 739-8526, Japan and 6Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo 113-8654, Japan Received June 10, 2014; Revised February 04, 2015; Accepted February 05, 2015 ABSTRACT understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes. The restriction-modification systems use epigenetic modification to distinguish between self and non- self DNA. A modification enzyme transfers a methyl INTRODUCTION group to a base in a specific DNA sequence while Restriction-modification (RM) systems recognize and at- its cognate restriction enzyme introduces breaks in tack nonself DNA based on DNA chemical modifications DNA lacking this methyl group. So far, all the restric- (Figure 1A). Among the various types of chemical mod- tion enzymes hydrolyze phosphodiester bonds link- ification, epigenetic methylation of bases is well studied. ing the monomer units of DNA. We recently reported Methylation occurs at specific bases, generating m5C (5- that a restriction enzyme (R.PabI) of the PabI super- methylcytosine, 5mC), m4C (N4-methylcytosine) or m6A family with half-pipe fold has DNA glycosylase activ- (N6-methyladenine, mA) at specific sequences (1). RM sys- ity that excises an adenine base in the recognition tems are frequently encountered in the prokaryotic world and, less often, in the eukaryotic world (REBASE, http: sequence (5 -GTAC). We now found a second activity / //rebase.neb.com)(2,3). These systems show mobility and in this enzyme: at the resulting apurinic apyrimidinic variability in sequence recognition (4) and interact in co- = (AP) (abasic) site (5 -GT#C, # AP), its AP lyase activ- operative or conflicting ways (5). RM systems have regula- ity generates an atypical strand break. Although the tory mechanisms reminiscent of mobile elements and toxin– lyase activity is weak and lacks sequence specificity, antitoxin systems (6). Bacterial strains can have very differ- its covalent DNA–R.PabI reaction intermediates can ent RM systems and methylomes, even genomic informa- be trapped by NaBH4 reduction. The base excision is tion indicates that they are closely related (7). not coupled with the strand breakage and yet causes The biological significance of RM systems is not yet fully restriction because the restriction enzyme action can understood. RM systems were discovered through host con- impair transformation ability of unmethylated DNA trolled variation of bacterial viruses: a virus propagated in even in the absence of strand breaks in vitro. The one host might not grow well in another host because of differences in DNA modification (8). RM systems attack base excision of R.PabI is inhibited by methylation of incoming DNA such as viral genomes and transforming the target adenine base. These findings expand our DNA as well as endogenous genomes with nonself epige- *To whom correspondence should be addressed. Tel: +81 3 5449 5326; Fax: +81 3 5449 5422; Email: [email protected] †These authors contributed equally to the paper as first authors. Present addresses: Masaki Fukuyo, Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan. Yoshikazu Furuta, Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Ken Ishikawa, Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702, USA. Miki Watanabe-Matsui, Department of Biochemistry, School of Medicine, Tohoku University, Sendai 980-8575, Japan. C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2842 Nucleic Acids Research, 2015, Vol. 43, No. 5 result suggested that DNA ends generated by R.PabI might be different from the 3-OH and 5-P ends. The product ends generated by purified R.PabI were difficult to religate, sug- gesting again that they did not have the typical 3-OH and 5-P end structures (Figure 1B (ii), see below). Our recent structural biology-based work demonstrated that R.PabI is a DNA glycosylase that excises an adenine base from the recognition sequence (Figure 1B (iii)) (20). This was unex- pected because base excision has often been associated with repair of DNA damages. It also reminds of excision of a Figure 1. Restriction enzymes. (A) Restriction-modification systems. For 5-methylcytosine base and its oxidized derivatives in DNA a specific DNA sequence, an enzyme makes epigenetic modifications such demethylation in plants and animals (21,22). as methylation to label self DNA. DNA without the modification is at- Finding of the base excision activity of the restriction en- tacked by the paired restriction enzyme. Duplex lines, double-stranded zyme immediately raises two questions. The first is about DNA. Box, specific sequence. Magenta diamond, chemical modification its relation to DNA strand breakage. The above work such as methylation (Me) at the recognition sequence. (B) Two possible / routes to DNA breakage for the restriction enzyme R.PabI. Top, a double- (20) concluded that the resulting AP (apurinic apyrimidinic stranded DNA with a recognition sequence (i). Left, hydrolysis of phos- = abasic) site is transformed into a strand break by the phodiester bonds to generate two 3-OH and 5-P ends (ii). Right, gener- ␤-elimination reaction at a high temperature in the (hy- ation of AP sites (iii). Cleavage generates two strand breaks (iv) with 5 -P per)thermophilic bacteria or by a separate AP endonuclease and 3-modified sugar (gray oval) ends. Red A, adenine to be excised unless methylated by a paired modification enzyme. See Supplementary Figure S4 in the mesophilic bacteria. A line of evidence for the latter for more detailed reaction mechanisms. route is the specific cleavage by the mixture of R.PabI and Escherichia coli lysate (20). This is consistent with specific cleavage with E. coli extract containing R.HpyAXII, a H. netic status (9,10). RM systems thus promote genetic isola- pylori homolog of R.PabI (19). The second question is its tion of a lineage. We hypothesize that they also drive adap- role in restriction. Which could be responsible for restric- tive evolution by introducing a specific global gene expres- tion phenomenon, base excision or strand breakage? sion pattern (7,11–12). In the present work, we addressed these questions. We All restriction enzymes examined so far hydrolyze phos- demonstrated a second activity in this enzyme that gener- phodiester bonds linking monomer nucleotide units leav- ates an atypical strand break at the AP site (Figure 1B(iv)). ing 3-OH (hydroxyl) and 5-P (phosphate) ends (Figure 1B This cleavage is, however, not always coupled to the base (i, ii)). Thus, they are phosphodiesterases. A break on one excision. The base excision reaction turned out to be suf- strand is often coupled with a nearby break on the comple- ficient for restriction because the enzyme action impaired mentary strand, leading to a double-strand break. Restric- DNA biological activity in the absence of strand breakage tion enzymes of Type II RM systems introduce a break at or in vitro. near a specific DNA sequence unless the sequence is methy- lated at a specific base (1). Type II restriction enzymes have been important for molecular biology and genetic engineer- MATERIALS AND METHODS ing (8). In addition, studies of their function and structure Bacterial strains, plasmids, viral genome and oligonucleotides
Load more

Recommended publications
  • 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]
  • Conventional Nzytech Restriction Enzymes: Activity Guide
    Conventional NZYTech Restriction Enzymes: Activity Guide Enzyme Activity in NZYTech buffers (%) Buffer Buffer for Restriction site Methylation Reaction Enzyme provided for double Isoschizomers (5’ →→→3’) A (green) B (blue) C (red) U (white) sensitive Conditions 100% activity digestion AscI GG ↓↓↓CGCGCC NZYBuffer C 80 100 100 100 NZYBuffer U * PalAI, SgsI (MB231) BamHI G↓↓↓GATCC NZYBuffer B 40 100 (<20) 100 NZYBuffer U * No None (MB064) BglII A↓↓↓GATCT NZYBuffer A 100 (100) (60) 100 NZYBuffer U * No None (MB065) CciNI (NotI) GC ↓↓↓GGCCGC NZYBuffer A 100 80 60 100 NZYBuffer U * NotI (MB153) DdeI C↓↓↓TNAG NZYBuffer U 25 25 25 100 NZYBuffer U No BstDEI, HpyF3I (MB236) MalI, BfuCI ♦♦♦, Bsp143I ♦♦♦, BstENII ♦♦♦, DpnI G(mA)↓↓↓TC NZYBuffer C 70 50 100 100 NZYBuffer U * (MB078) BstKTI ♦♦♦, BstMBI ♦♦♦, DpnII ♦♦♦, Kzo9I ♦♦♦, NdeII ♦♦♦ BfuCI, Bsp143I, BssMI, BstKTI, BstMBI, DpnII ↓↓↓GATC NZYBuffer C 80 80 100 100 NZYBuffer U * (MB233) Kzo9I, MboI, NdeII, Sau3AI EcoRI G↓↓↓AATTC NZYBuffer A 100 (120) (80) 100 NZYBuffer U * FunII (MB067) EcoRV GAT ↓↓↓ATC NZYBuffer A 100 0 25-50 100 NZYBuffer U * Eco32I (MB068) HindIII A↓↓↓AGCTT NZYBuffer B <20 100 (100) 100 NZYBuffer U * No None (MB070) HinfI G↓↓↓ANTC NZYBuffer C (90) (90) 100 100 NZYBuffer U * None (MB239) HpaI GTT ↓↓↓AAC NZYBuffer B 20 100 (80) 100 NZYBuffer U * KspAI (MB071) KpnI GGTAC ↓↓↓C NZYBuffer C 50 (80) 100 100 NZYBuffer U * No Acc65I ♦♦♦, Asp718I ♦♦♦ (MB072) BfuCI, Bsp143I, BstENII, BstMBI, DpnII, MboI ↓↓↓GATC NZYBuffer C 100 100 100 100 NZYBuffer U * (MB241) Kzo9II, NdeII, BstKTI ♦♦♦ MluI
    [Show full text]
  • Curriculum Vitae SIR RICHARD JOHN ROBERTS ADDRESS PERSONAL
    Curriculum Vitae SIR RICHARD JOHN ROBERTS ADDRESS New England Biolabs 240 County Road, Ipswich, MA 02138 USA Email: [email protected] Telephone: (978) 380-7405 / Fax: (978) 380-7406 PERSONAL Born on September 6, 1943, Derby, England EDUCATION 1962-1965 University of Sheffield, Sheffield, England B.Sc. in Chemistry 1966-1968 University of Sheffield, Sheffield, England Ph.D. in Organic Chemistry POSITIONS 2005- Chief Scientific Officer, New England Biolabs 1992-2005 Research Director, New England Biolabs 1986-92 Assistant Director for Research, Cold Spring Harbor Laboratory 1972-86 Senior Staff Investigator, Cold Spring Harbor Laboratory 1971-1972 Research Associate in Biochemistry, Harvard University 1969-1970 Research Fellow, Harvard University OUTSIDE ACTIVITIES 1974-1992 Consultant and Chairman of Scientific Advisory Board New England Biolabs 1977-1985 Scientific Advisory Board, Genex Corp. 1977-1987 Editorial Board: Nucleic Acids Research 1979-1984 Editorial Board: Journal of Biological Chemistry 1982-1989 Member: National Advisory Committee of GENBANK 1984-1986 Member: National Advisory Committee of BIONET 1985-1988 Panel member: NIH Study Section in Biochemistry. 1985-2002 Editorial Board: Bioinformatics (formerly CABIOS) 1987-1990 Chairman: National Advisory Committee of BIONET 1987-2009 Senior Executive Editor: Nucleic Acids Research 1990-1992 Panel member: NCI Cancer Centers Support Grant Review Committee 1993-1995 Panel member: NLM Study Section/Comp. Biol. 1994-2000 Scientific Advisory Board, Molecular Tool 1994- Patron of the Oxford International Biomedical Center 1996-1998 Visiting Professor, University of Bath, UK. 1996-2000 Chairman, NCI Board of Scientific Counselors 1996-1999 Scientific Advisory Board, Oxford Molecular Group 1997-2001 Editorial Board: Current Opinion Chem. Biol.
    [Show full text]
  • Restriction Enzymes Are Molecular Scissors
    Molecular Scissors Restriction enzymes are molecular scissors DR. ABUL HASAN SARDAR Assistant Professor and Head Department of Microbiology Sarsuna College RESTRICTION ENZYMES • A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double- stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites. Property of restriction enzymes • They that link adjacent nucleotides in DNA molecules. HOW RESTRICTION ENZYMES WORKS? • Restriction enzymes recognize a specific sequence of nucleotides, and produce a double-stranded cut in the DNA, these cuts are of two types: • BLUNT ENDS. • STICKY ENDS. Blunt end Sticky end BLUNT ENDS • These blunt ended fragments can be joined to any other DNA fragment with blunt ends. • Enzymes useful for certain types of DNA cloning experiments “STICKY ENDS” ARE USEFUL DNA fragments with complimentary sticky ends can be combined to create new molecules which allows the creation and manipulation of DNA sequences from different sources. • While recognition sequences vary widely , with lengths between 4 and 8 nucleotides, many of them are palindromic. PALINDROMES IN DNA SEQUENCES Genetic palindromes are similar to verbal palindromes. A palindromic sequence in DNA is one in which the 5’ to 3’ base pair sequence is identical on both strands (the 5’ and 3’ ends refers to the chemical structure of the DNA). PALINDROME SEQUENCES • The mirror like palindrome in which the same forward and backwards are on a single strand of DNA strand, as in GTAATG • The Inverted repeat palindromes is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (GTATAC being complementary to CATATG) • Inverted repeat palindromes are more common and have greater biological importance than mirror- like palindromes.
    [Show 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.
    [Show full text]
  • The Mmei Family: Type II Restriction–Modification Enzymes That Employ Single-Strand Modification for Host Protection Richard D
    5208–5221 Nucleic Acids Research, 2009, Vol. 37, No. 15 Published online 3 July 2009 doi:10.1093/nar/gkp534 The MmeI family: type II restriction–modification enzymes that employ single-strand modification for host protection Richard D. Morgan*, Elizabeth A. Dwinell, Tanya K. Bhatia, Elizabeth M. Lang and Yvette A. Luyten New England Biolabs, Inc. 240 County Road Ipswich, MA 01938 USA Received March 18, 2009; Revised and Accepted June 8, 2009 ABSTRACT material between ‘self’ and ‘others’ within microbial popu- lations (2). As such they represent fertile ground for the The type II restriction endonucleases form one of study of protein evolution, particularly the evolution the largest families of biochemically-characterized of those protein–DNA interactions that confer sequence proteins. These endonucleases typically share little specificity to these enzymes. sequence similarity, except among isoschizomers A restriction system must differentiate between ‘self’ that recognize the same sequence. MmeI is an unu- and ‘foreign’ DNA in order to cut the identifiably foreign sual type II restriction endonuclease that combines DNA but not host DNA. This discrimination is based endonuclease and methyltransferase activities in a upon modification of the DNA, usually in the form of single polypeptide. MmeI cuts DNA 20 bases from methylation of a base in each strand within the discrete its recognition sequence and modifies just one DNA DNA sequence recognized by the endonuclease. Usually, strand for host protection. Using MmeI as query we hemi-methylated DNA is not cut by the type II endonu- have identified numerous putative genes highly sim- cleases, ensuring that immediately following replication the hemi-methylated daughter chromosomes remain pro- ilar to MmeI in database sequences.
    [Show full text]
  • Infectious RNA Transcripts from Full-Length Dengue Virus Type 2 Cdna Clones Made in Yeast
    JOURNAL OF VIROLOGY, July 1997, p. 5366–5374 Vol. 71, No. 7 0022-538X/97/$04.0010 Copyright © 1997, American Society for Microbiology Infectious RNA Transcripts from Full-Length Dengue Virus Type 2 cDNA Clones Made in Yeast 1 2 1 1 STEPHANIE POLO, GARY KETNER, ROBIN LEVIS, AND BARRY FALGOUT * Laboratory of Vector-Borne Viral Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda,1 and Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health, Baltimore,2 Maryland Received 23 December 1996/Accepted 18 March 1997 The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The full- length dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E. coli shuttle vector. Full-length cDNA clones were propagated once in E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK2 cells produced typical dengue infection, as judged by cytopathic effects and indirect immunofluorescence. Infectivity was sensitive to RNase digestion and was dependent on the presence of cap analog in the transcription reaction mixture. Virus in the medium was passaged on C6-36 cells to produce stocks, and these stocks had titers and plaque morphologies similar to those of the parental dengue virus type 2. Intracellular dengue virus RNA from cells infected with transcript- derived virus contained an introduced BstEII site, proving that infectivity was derived from RNA transcripts and not from contamination with parental dengue virus.
    [Show full text]
  • Assignment 1 Part I
    1 Assignment 1 Note: Part I of the assignment is to be submitted through Blackboard (See instructions on Blackboard). Parts II, III and IV are to be submitted as a hard copy during your lab session. Part I Dilutions and Concentrations When doing your calculations, do not round off your intermediate numbers. Only round off the final answer. Your answers must be submitted to two significant figures after the decimal. For example 2.00, 0.020, or 0.0020. It is strongly recommended that you submit your answers using the web browser Firefox. You will be allowed two submissions! Use the following information to answer questions 1-6 You prepare a solution by adding the following ingredients in the order indicated: 600 mL H2O, 125 mL 1.6 M LiCl, 50 mL 20 % (m/v) MgCl2, and 25 mL 10g/L NaCl. The properties of each ingredient are as follows: LiCl: MW 200g/mole, density 1.2g/mL, density of 1.6 M soln. 1.05g/mL MgCl2: MW 150g/mole, density 1.3g/mL, density of 20% soln. 1.1g/mL NaCl: MW 35g/mole, density 1.15g/mL, density of 10g/L soln. 1.03g/mL Final solution: density: 1.25g/mL 1. What is the final molarity of MgCl2 in the solution? (0.083M) 2. What is the volume in milliliters of one part? (25 mL) 3. What is the percentage (m/m) of NaCl in the final solution? (0.025%) 4. What is the percentage (m/v) of LiCl in the final solution? (5%) 5. What is the number of parts of solvent in the final solution? (24 parts) 6.
    [Show full text]
  • R.Sspd5i Is a Neoschizomer of Hphi Producing Blunt End DNA Fragments
    FEBS Letters 448 (1999) 38^40 FEBS 21798 R.SspD5I is a neoschizomer of HphI producing blunt end DNA fragments Lyudmila Zheleznayaa, Sergey Shiryaevb, Elena Zheleznyakovab, Nadezhda Matvienkoc, Nicholas Matvienkob;* aInstitute of Theoretical and Applied Biophysics, RAS, 142292 Pushchino, Russia bInstitute of Protein Research, RAS, 142292 Pushchino, Russia cBranch of the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 142292 Pushchino, Russia Received 18 February 1999 reaction bu¡er (10 mM Tris-HCl, pH 7.4, 100 Wg/ml albumin, 10 mM Abstract The strain Staphylococcus species D5 produces a MgCl2) were 10, 50 and 100 mM for LRB, MRB and HRB, respec- restriction enzyme. It is the neoschizomer of HphI endonuclease, tively. The pH values of the reaction bu¡er were 7.0, 7.4, 8.0. Incu- which cleaves DNA at a distance of eight nucleotides from the bation temperatures were 28, 37 and 48³C. recognition sequences producing blunt end DNA fragments: 5P- GGTGA8Ns-3P and 3P-CCACT8Nu-5P. 2.3. Determination of DNA cleavage points z 1999 Federation of European Biochemical Societies. Cleavage points on DNA were determined by the elongated primer method [6]. As a template, we used for the ¢rst time the recombinant phage DNAs specially constructed for this study (M13tg130: Key words: Site-speci¢c endonuclease; Neoschizomer; VCIHindIII and M13tg131: VCIHindIII) containing the small frag- Restriction ment of VCI857S7 DNA with coordinates 37459^37584 inserted in opposite orientations. These phage DNAs contained the recognition site for the SspD5I endonuclease near the universal primer. To make DNA recombinants containing multiple recognition sites 1. Introduction for the endonuclease SspD5I in di¡erent surrounding nucleotide se- quences, we used a synthetic oligonucleotide: 5P-AGATCT11NT- Site-speci¢c endonucleases are in the forefront of the en- CACC3NGGATCC-3P, which contains the recognition site for zymes used in genetic engineering.
    [Show full text]
  • Restriction-Enzymes
    Methods for cutting DNA Chemical methods Mechanical shearing in a blender Restriction Endonuclease digestion sonication Restriction and Modification systems: reported in several bacteria s Function: Restriction systems checks the presence of foreign DNA and destroys it. s recognize specific sequences in the incoming DNA and breaks into fragments. s Cutting is either at specific sites or random. Discovery s Bacteriophages: have a single host they can infect, or a small number of related bacteria: which is the ‘host range’ s This effect is seen in efficiency of plating, i.e. the number of plaques formed in plating tests gets reduced. s Observation: In the 50’s and 60’s occasionally a virus could become active in a new strain but would no longer be effective in the original host Discovery s This change in host range seemed to be associated with specific bases in the viral DNA being methylated: s This is called modification : a change conferred by the host Methylases s 1969 Werner Arber and Stewart Linn discovered that most bacteria contained a class of enzymes which modify the bases in DNA termed methylases. s These enzymes recognize a specific sequence of DNA and add a methyl group onto certain bases, most often an adenine or cytosine. s The presence of these methyl groups on the viral DNA seemed to be the cause of their ability to grow in some strains of bacteria, but not others. methylation effect: s This ability to grow was because the methyl groups were protecting the viral DNA from an unusual type of enzyme.
    [Show full text]
  • (I /1 Alan Grodzinsky
    Analysis of the Structural Changes Caused by Positive DNA Supercoiling by- Marita Christine Barth B.S. Bioresource Research Oregon State University, 1998 Submitted to the Division of Biological Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Macromolecular Biochemistry and Biophysics at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY February 2007 C 2007 Massachusetts Institute of Technology. All rights reserved. Signature of Author: Division of Biological Engineering January 12, 2007 Certified by: (I Peter C. Dedon Professor of Toxicology and Biological Engineering Thesis Supervisor Accepted by: L". % SLr/ .,: Alan Grodzinsky Irof's r of Biological Engineering rMASSACK·KIEMEWIJf nNte OF TECHNOLOGY /1 Chairman, Co ittee for Graduate Students AUG 0 2 2007I LIBRARIES ARQHNi This doctoral thesis has been examined by a committee of the Division of Biological Engineering as follows: Associate Professor Bevin P. Engelward Chairman Professor Peter C. Dedon Supervisor Professor John M. Essigmann fl,(\ Dr. Richard J. Roberts --- Analysis of the Structural Changes Caused by Positive DNA Supercoiling by Marita Christine Barth Submitted to the Division of Biological Engineering on January 12, 2007 in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Macromolecular Biochemistry and Biophysics ABSTRACT The procession of helix-tracking enzymes along a DNA molecule results in the formation of supercoils in the DNA, with positive supercoiling (overwinding) generated ahead of the enzyme, and negative supercoiling (underwinding) in its wake. While the structural and physiological consequences of negative supercoiling have been well studied, technical challenges have prevented extensive examination of positively supercoiled DNA. Studies suggest that at sufficiently high levels of overwinding, DNA relieves strain by adopting an elongated structure, where the bases are positioned extrahelically and the backbones occupy the center of the helix.
    [Show full text]
  • Assignment 1 Part I
    1 Assignment 1 Note: Part I of the assignment is to be submitted through Blackboard (See instructions on Blackboard). Parts II, III and IV are to be submitted as a hard copy during your lab session. Part I Dilutions and Concentrations When doing your calculations, do not round off your intermediate numbers. Only round off the final answer. Your answers must be submitted to two significant figures after the decimal. For example 2.00, 0.020, or 0.0020. It is strongly recommended that you submit your answers using the web browser Firefox. You will be allowed two submissions! Use the following information to answer questions 1-6 You prepare a solution by adding the following ingredients in the order indicated: 600 mL H2O, 125 mL 1.6 M LiCl, 50 mL 20 % (m/v) MgCl2, and 25 mL 10g/L NaCl. The properties of each ingredient are as follows: LiCl: MW 200g/mole, density 1.2g/mL, density of 1.6 M soln. 1.05g/mL MgCl2: MW 150g/mole, density 1.3g/mL, density of 20% soln. 1.1g/mL NaCl: MW 35g/mole, density 1.15g/mL, density of 10g/L soln. 1.03g/mL Final solution: density: 1.25g/mL 1. What is the final molarity of MgCl2 in the solution? (0.083M) 2. What is the volume in milliliters of one part? (25 mL) 3. What is the percentage (m/m) of NaCl in the final solution? (0.025%) 4. What is the percentage (m/v) of LiCl in the final solution? (5%) 5. What is the number of parts of solvent in the final solution? (24 parts) 6.
    [Show full text]