Site pages
Current course
Participants
General
20 February - 26 February
27 February - 5 March
6 March - 12 March
13 March - 19 March
20 March - 26 March
27 March - 2 April
3 April - 9 April
10 April - 16 April
17 April - 23 April
24 April - 30 April
Lesson 10. RESTRICTION ENZYMES
Module 3. Genetic engineering technology / recombinant DNA technology
Lesson 10
RESTRICTION ENZYMES
10.1 Restriction Modification (RM) System in BacteriaRESTRICTION ENZYMES
Like the immune system which operates in eukaryotes for their protection against foreign agents /antigens, Restriction Modification (called RM) system operates in bacteria for destroying foreign DNA. This phenomenon was first discovered when bacteriophages were transferred from one bacterium to another. Bacteria have developed inbuilt defense mechanisms to protect themselves from foreign invasion. The RM system comprises of a restriction endonuclease and a methylase as shown in Fig. 10.1. Restriction enzymes are unable to digest the host DNA since they methylate it.
Restriction enzymes cut the DNA sequence at a particular site whereas methylases add up a methyl group at that site in the DNA sequence recognized by the restriction enzymes and thus, that methylated DNA sequence can not be cleaved by the restriction endonucleases. The RM phenomenon is elaborated in Fig. 10.2. If a bacteriophage is grown in an E. coli strain B and then titred on E. coli B and K strains, the phage will be restricted by strain K. However, any particle of bacteriophage which results from infection of E. coli strain K, is retitred on E. coli strain K, it will not be restricted as it has been methylated by E. coli K. The restrictive host strain protects its own DNA from restriction endonuclease by modifying the same appropriately.
10.2 Restriction Enzymes
Restriction endonucleases (RE) are the biological scissors naturally present in bacteria that cleave the incoming foreign DNA entering therein. The breakthrough in restriction endonucleases came in 1970 with the discovery of the first RE enzyme HindII from Haemophilus influenzae for which Daniel Nathans, Werner Arber and Hemilton Smith got nobel prize in 1978. The discovery of Restriction enzymes was the turning point in Molecular Biology that led to the development of Recombinant DNA technology and the very first use of RE was made in the production of human insulin in E. coli. Restriction endonucleases are DNA cutting enzymes which recognize and cut DNA only at specific sequence of nucleotides. These are four types of restriction endonucleases namely Type I, II, III and IV as described below:
10.2.1 Type I enzymes
Type I restriction enzymes are REs whose cleavage site is 1000 bp away from the 5’ end of “TCA” located in recognition site and the recognition site is 15 bp in length. These enzymes require magnesium ions (Mg2+), S-adenosyl methionine (S-adomet) and hydrolyzed adenosine triphosphate (ATP) for restriction. The examples are EcoB and EcoK. Their recognition sequences are given below:
• EcoK : 5’ AAC(N)8GTGC 3’
• EcoB : 5’ TGA(N)8TGCT 3’
• EcoB : 5’ TGA(N)8TGCT 3’
10.2.2 Type II enzymes
Type II REs recognize a particular (specific) target sequence in a duplex DNA molecule and cut polynucleotide chains within that sequence to give rise to discrete staggered / cohesive or blunt DNA fragments of defined length and sequence. Type II REs are the most extensively used endonucleases and constitute the most ideal tools for molecular cloning and require only Mg2+ ions as co-factor. Examples include EcoRI, BamHI, HindIII, PstI, NotI etc.
10.2.3 Type III enzymes
Like Type1 REs, recognition site for type III is specific but not always at a symmetric sequence and cleavage site is at a specific distance from recognition site i.e. in the immediate vicinity of recognition site. These enzymes require magnesium ions (Mg2+), S-adenosyl methionine (S-adomet) and hydrolyzed adenosine triphosphate (ATP) for restriction. The example is of HgaI whose recognition sequence is 5’GACGC3’ but cleavage site is 5-10 bases away from it.
10.2.4 Type IV enzymes
These REs are modification dependent restriction enzymes and are composed of proteins that cut only methylated DNA.
The differences between three types of restriction enzymes Type I, II and III are given in Table 10.1.
Out of the four types of restriction enzymes, the type II restriction enzymes are useful for molecular cloning and hence will be dealt in detail in the following section:
Table 10.1. Differences between Type I, II and III Restriction Endonucleases
10.3 Type II Restriction Enzymes
Type II restriction endonucleases are extensively used biological tools in molecular cloning experiments. Type II restriction endonucleases have been divided into two categories i.e. Type IIa and Type IIb as per their end specificity.
10.3.1 Type IIa
The type IIa REs cleave the DNA sequence within the recognition site in a staggered pattern generating specific fragments with identical single stranded cohesive / sticky / staggered 5’ to 3’ (EcoRI) as shown in Fig. 10.3 and 3’ to 5’ ends (PstI) as follows in Fig. 10.4.
Fig. 10.4 Recognition sequence and cleavage of DNA with PstI
10.3.2 Type IIb (Blunt ends)
The type IIa REs generates flush / blunt ends e.g. HindII, SmaI as shown in Fig. 10.5.
Fig. 10.5 Recognition sequence and cleavage of DNA with SmaI
10.4 Restriction Enzyme Sites for Some Common REs
The following Table 10.2 gives recognition sequences and cleavage sites for some commonly used REs.
Table 10.2 Restriction enzyme sites for some common type II enzymes
10.5 Frequency of Cutting of Recognition Enzymes
Based on cleavage, enzymes are recognized as Tetra, Penta, Hexa, Octa cutters. The frequency of cutting is calculated as 1/4n (n = no. of nucleotides). Some examples are:
Sau 3A (GATC) cuts 1/44 i.e. (¼)(¼)(¼)(¼) = once every 256 bp
BamH1 (GGATCC) cuts 1/46 i.e. (¼)(¼)(¼)(¼)(¼)(¼) = once every 4096 bp
BamH1 (GGATCC) cuts 1/46 i.e. (¼)(¼)(¼)(¼)(¼)(¼) = once every 4096 bp
10.6 Nomenclature of Restriction Enzymes
The nomenclature of restriction enzymes was given by Smith and Nathans in 1973. The first three letters of REs represent first letter of genus and two letters of species name of the organism from which these enzymes were isolated e.g. Eco from Escherichia coli, Hin from Haemophilus influenzae. Fourth letter represents serotype of the organism e.g. EcoR where R is serotype and Hinf where f is the serotype. The last Roman letter is for number of enzymes recovered from the same organism e.g. EcoRI, EcoRII means two different REs from E. coli were isolated.
Example of EcoRI
E Escherichia (genus)
co coli (species)
R RY13 (strain)
I First identified in E. coli
co coli (species)
R RY13 (strain)
I First identified in E. coli
10.7 Isoschizomers
The following types of enzymes are known as Isochizomers.
i) REs which share the same recognition and cleavage sites
-HindIII and HsuI where HindIII is from Haemophilus influenzae Rd and HsuI is from Haemophilus suis
MboI also cleaves at /GATC, and so is an isoschizomer of Sau3A (/GATC)
ii) REs which share same recognition sequence but differ in cleavage sites
– SmaI and XmaI
iv) Some REs recognize only unmethylated sequences, their isoschizomers cleave both methylated and unmethylated sites
The modification enzymes are designated as Methylases and they recognize the same DNA sequences as REs. However, the modification enzymes are differentiated from REs by writing letter M before RE. They modify the DNA by methylating either adenine at 6N position or cytosine at 5C position and require Mg2+, ATP and S -adomet (as methyl donor). Based on methylation pattern, they are designated as ‘dam’ and ‘dcm’ methylases. The ‘dam’ methylase adds methyl group at 6N adenine e.g. 5’G*ATC3’. The susceptible RE sites include DpnI, PvuI,BclI, BglII, Sau3A, MboI etc. On the other hand, ‘dcm’ methylase adds methyl groups at 5C cytosine 5’C*CAGG or C*CTGG 3’ (EcoRII). The susceptible RE sites include BamHI, BalI, KpnI, NarI etc.
10.9 Application of Restriction Enzymes
10.9.1 Molecular cloning/ recombinant DNA technology
Molecular cloning involves amplification of a gene of interest from the target and digesting it with restriction enzymes for ligation into vector cut with same enzymes to generate recombinant gene for introduction into prokaryotic or eukaryotic host. It has already been discussed in detail in previous section.
10.9.2 Restriction digestion
The process of cutting/digesting DNA molecules into smaller pieces with REs is known as restriction digestion. Reactions are set up and samples are electrophoresed to check for the generation of restriction fragments. Some of the REs are influenced by reaction conditions and exhibit star activity e. g Eco Star Activity. The star activity is exhibited due to deviations from optimal reaction conditions which alter the cleavage specificity. In buffers containing 100 mM NaCl, 5mM MgCl2, pH 7.3, EcoRI recognizes and cleaves the sequence GAATTC. However, increasing the pH, lowering salt concentration, replacing magnesium by manganese etc. alter the specificity of EcoRI. EcoRI then recognizes AATT which occurs more frequently and starts cutting non-specifically. This new specificity is called EcoRI* i.e. star activity.
10.9.3 Restriction mapping
Restriction Enzymes are also used to map the location of restriction enzyme sites on vector or any gene.
Besides above, the other applications of REs include DNA Sequencing, RFLP (Restriction fragment length polymorphism), DNA Finger printing, Epidemiological and hybridization studies.
Books
Molecular Biotechnology - Second Edition, S. B. Primrose, Blackwell Science Inc., ASIN: 0632030534
Introduction to Biotechnology, Brown, C.M., Campbell, I and Priest, F.G. Panima Publishing Corporation, 2005. ISBN : 81-86535-42-X
DNA and Biotechnology, Fitzgerald-Hayes, M. And Reichsman, F. 2nd Amsterdam : Elsevier, 2010. ISBN : 0-12-048930-5
Molecular Biotechnology : Principles and Applications of Recombinant DNA, Glick, B.R., Pasternack, Jack, J and Patten, Cheryl, L (Eds)., 4th Washington., ASM Press, 2010. ISBN : 1-55581-498-4
Molecular Biology and Biotechnology : a guide for students, Krauzer, H. And Massey, A.(Eds) 3rd Washington DC : ASM Press, 2008, ISBN : 978-155581-4724
Recombinant DNA and Biotechnology : a guide for teachers, Kreuzer, H and Massey, A. 2nd Washington : ASM Press, 2001, ISBN : 155581-175-2
Molecular Biotechnology, Primrose, S.B. 2nd New Delhi : Panima, 2001. ISBN : 81-86535-21-7
Molecular Biology and Biotechnology, Smith, C.A. and Wood, E.J. London : Chapman and Hall, 1991.ISBN : 0-412-40750-7
Introduction to Biotechnology, Thieman, W.J and Pallidino, M.A. 2nd New York : Pearson, 2009, ISBN : 978-0-321-58903-3
Molecular Biology and Biotechnology, Walker, J.M and Rapley, R. 4th – New Delhi : Panima Publishing Corporation, 2003, ISBN : 81-86535-40-3
Gene Biotechnology, Wu William, Welsh, M.J., Kaufman, P.B and Zhang, H.H. 2nd Boca Raton : CRC press, 2004. ISBN : 0-8493-1288-4
From Genes to Genomes: Concepts and Applications of DNA Technology, 3rd Edition, Jeremy W. Dale, Malcolm von Schantz, Nicholas Plant (Eds), Wiley-Blackwell, 2011, ISBN: 978-0-470-68386-6
Microbial Genetics, 2nd Edition, Stanly R Maloy, John Cronan, David Freifelder, Narosa, ISBN: 8173196974
Molecular Biology of the Gene, Sixth Edition, James D. Watson (Editor) Cold Spring Harbour Press and Benjamin Cummings, ISBN 978-080539592-1
Internet Resources
http://en.wikipedia.org/wiki/Restriction_enzyme
www.dnalc.org/resources/animations/restriction.html
www.youtube.com/watch?v=-sI5vy-cD2g
http://escience.ws/b572/L5/L5.htm
http://www.scq.ubc.ca/restriction-endonucleases-molecular-scissors-for-specifically-cutting-dna/
Last modified: Thursday, 1 November 2012, 5:48 AM