the isolation of the restriction endonuclease from E coli K Meselson amp Yuan 1968 It was evident that the restriction endonucleases from E coli K and E coli B were important examples of proteins ID: 1009015
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1. Restriction Enzymes.3
2. The biochemistry of restriction advanced withthe isolation of the restriction endonuclease fromE. coli K (Meselson & Yuan 1968). It was evident that the restriction endonucleases from E. coli K and E. coli B were important examples of proteinsthat recognize specific structures in DNA, but theproperties of these type I enzymes as they are nowknown, were complex.Discovery of a restriction endonuclease (HindII) fromHaemophilus influenzae strain Rd (Smith & Wilcox1970) and the elucidation of the nucleotidesequence at its cleavage sites in phage T7 DNA.
3. Hamilton O. Smith discovered and isolated the first site-specific restriction endonuclease HindII from the bacterium Haemophilus influenzae.HindII: The first Type II REase discovered was HindII from the bacterium Haemophilus influenzae Rd. (1970)EcoRI was soon discovered (1972). They were rapidly exploited in the first recombinant DNA experiments.
4. The Nobel Prize in Physiology or Medicine 1978 was awarded jointly to Werner Arber, Daniel Nathans and Hamilton O. Smith "for the discovery of restriction enzymes and their application to problems of molecular genetics."https://www.nobelprize.org/prizes/medicine/1978/summary/
5. Types of restriction and modification(R-M) systemThere are four categories of restriction modification systems: type I, type II, type III and type IV, all with restriction enzyme activity and a methylase activity (except for type IV that has no methylase activity). They were named in the order of discovery, although the type II system is the most common.
6. Type IOne enzyme with different subunits for recognition, cleavage and methylation.Oligomeric REase and MTase complexRequire both ATP and S-adenosyl-L-methionine to functionCleave variably, often far from recognition site (cleaves DNA up to 1000 bp away)little value for gene manipulationType I systems are the most complex, consisting of three polypeptides: R (restriction), M (modification), and S (specificity). The resulting complex can both cleave and methylate DNA. Both reactions require ATP, and cleavage often occurs a considerable distance from the recognition site. The S subunit determines the specificity of both restriction and methylation. Cleavage occurs at variable distances from the recognition sequence, so discrete bands are not easily visualized by gel electrophoresis.These features mean that type I systems are of little value for gene manipulation.Example: EcoK1Genes: hsdR, hsdM, hsdS
7. Type IITwo different enzymes which both recognize the same target sequence, which is symmetrical. The two enzymes either cleave or modify the recognition sequence.Separate REase and MTase or combined REase∼MTase fusionCleave within or at fixed positions close to recognition siteType II restriction enzymes cleave within the recognition site itself or at a closer distance to it.They do not require adenosine triphosphate (ATP) for DNA cleavage ; most require magnesiumMany different subtypesUseful in gene manipulationExample: EcoRIGenes: ecorIR, ecorIM
8. Type IIIOne enzyme with two different subunits, one for recognition and modification, and one for cleavage.Combined REase + MTase complexATP required for restrictionS-adenosyl-L-methionine stimulates reaction but is not requiredCleave at fixed position outside recognition site (24-26 bp away from site)little value for gene manipulationExample: EcoP1Genes: ecoP1IM, ecoP1IR
9. Type I: Bi-functional enzyme , Three different subunitsType II: Uni-functional enzyme, Two identical subunitsType III: Bi-functional enzyme , Two different subunits
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11. Type IVMethylation-dependent REase The modification-dependent Type IV enzymes are highly diverseCleave at variable distance from recognition siteCleave m6A, m5C, hm5C and/or other modified DNAMany different typesm6A: N6-methyladeninem5C: 5-methylcytosinehm5C: 5-hydroxymethylcytosineExample: EcoKMcrAGenes: mcrA, mcrBC, mrr Type IV systems are not true RM systems because they only contain a restriction enzyme and not a methylase. Unlike the other types, type IV restriction enzymes recognize and cut only modified DNA (for example methylated DNA)
12. Some viruses have evolved ways of subverting the restriction modification system, usually by modifying their own DNA, by adding methyl or glycosyl groups to it, thus blocking the restriction enzymes. Other viruses, such as bacteriophages T3 and T7, encode proteins that inhibit the restriction enzymes.To counteract these viruses, some bacteria have evolved restriction systems which only recognize and cleave modified DNA, but do not act upon the host's unmodified DNA. Evolutionary tussle
13. Where it is necessary to distinguish between therestriction and methylating activities, they are giventhe prefixes ‘R’ and ‘M’, respectively, e.g. R.SmaI andM.SmaI.
14. Examples of restriction endonuclease nomenclature.Enzyme Enzyme source Recognition sequenceSmaI Serratia marcescens, 1st enzyme CCCGGGHaeIII Haemophilus aegyptius, 3rd enzyme GGCCHindII Haemophilus influenzae, strain d, 2nd enzyme GTPyPuACHindIII Haemophilus influenzae, strain d, 3rd enzyme AAGCTTBamHI Bacillus amyloliquefaciens, strain H, 1st enzyme GGATCC
15. Restriction Sites or Recognition SequencesType II:Restriction endonucleases recognize specific sequences 4 to 8 base pairs long. These restriction sites are typically pallindromic - they read identically on both strands.The dyad symmetry of restriction sites suggests that restriction enzymes act as dimers - each subunit interacting with one half of the site. This has been confirmed for EcoRI.Mechanistically, restriction enzymes are believed to bind non-specifically to the sugar-phosphate backbone of the target molecule. The enzyme can then use the backbone as a track to scan the DNA for an appropriate sequence by probing for sequence-specific features in the major groove of the molecule. On finding such a site, the enzyme binds, and in the presence of Mg+2 undergoes a conformational change which kinks the helix and breaks the backbone of each strand to produce fragments with 5' phosphate and 3' hydroxyl groups.
16. 5′-G/AA* T T C-3′3′-C TT A*A/G-5′The position at which the restricting enzyme cuts isusually shown by the symbol ‘/’ and the nucleotidesmethylated by the modification enzyme are usuallymarked with an asterisk.G/AATTCDepending on where in the recognition site relative to the axis of dyad symmetry the enzyme cleaves the DNA strands, three types of ends are produced.1. If the enzyme cuts at the axis, blunt ends are produced.2. If the enzyme cuts to the left of the axis, 5' overhangs are produced.3. If the enyzme cuts to the right of the axis, 3' overhands are produced.1.2.3.
17. One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl.Reaction: RE DNA BufferSuitable TemperatureAll can be inactivated at high temperature catalyzes the hydrolysis of the phosphodiester bond between adjacent nucleotides.For example, the restriction endonuclease EcoRI has the EC number EC 3.1.23.13Lab
18. Sticky or Cohesive endsBlunt or flush ends
19. A straight cut of restriction enzymes generates blunt ends, where both strands terminate in a base pair. Blunt ends are also called non-cohesive ends, since there is no unpaired DNA strand fleeting at the end of DNA. The sticky ends, a.k.a. cohesive ends, have unpaired DNA nucleotides on either 5’- or 3’- strand, which are known as overhangs. These overhangs are most often generated by a staggered cut of restriction enzymes. Sticky ends are generally more desired in cloning technology where a DNA ligase is used to join two DNA fragments into one, because the yield and specificity of ligation using sticky ends is significantly higher that with blunt ends.
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22. The same sticky ends will be produced by different endonuclaesesSuch as by the Sau3A and BamHI
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24. 1. Isoschizomers are pairs of restriction enzymes specific to the same recognition sequence. For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other.The first enzyme discovered which recognizes a given sequence is known as the prototype; all subsequently identified enzymes that recognize that sequence are isoschizomers. Isoschizomers are isolated from different strains of bacteria and therefore may require different reaction conditions.Different Enzymes/Same Recognition Site/Same Cut
25. 2. An enzyme that recognizes the same sequence but cuts it differently is a neoschizomer. Neoshizomers are enzymes that recognize the same sequence, but cut it differently.Different Enzymes/Same Recognition Site/different CuttingFor example, SmaI (CCC/GGG) and XmaI (C/CCGGG) are neoschizomers of each other. Similarly Kpn1 (GGTAC/C) and Acc651 (G/GTACC) are neoschizomers of each other.
26. 3. An enzyme that recognizes a slightly different sequence, but produces the same ends is an isocaudomer.Different Enzymes/different Recognition Site/different Cutting/ producing same endsUpon cleavage of DNA, generate identical overhanging termini sequences
27. 4-mer sequence will occur1/4 x 1/4 x 1/4 x 1/4= 1/256 bp6-mer sequence will occur1/4 x 1/4 x 1/4 x 1/4 x 1/4 x 1/4= 1/4096 bp8-mer sequence will occur1/4 x 1/4 x 1/4 x 1/4 x 1/4 x 1/4 x 1/4 x 1/4= 1/65536 bpFrequency of Restriction Enzyme Sites
28. A restriction map is a map of known restriction sites within a sequence of DNA. Restriction mapping requires the use of restriction enzymes.
29. 1. In bacteria, the restriction endonuclease provides a defense against foreign DNA, such as that borne by bacteriophages. But how bacteria are able to protect their own DNA?(A) By phosphorylation of bacterial DNA by restriction enzyme(B) By methylation of foreign DNA by restriction enzyme(C) By phosphorylation of foreign DNA by restriction enzyme(D) By methylation of bacterial DNA by restriction enzyme2. Which type of restriction endonucleases is used most in genetic engineering?(A) Type I(B) Type II(C) Type III(D) Type IV3. Find the microorganism that can be the source of the restriction endonuclease AluI (A) Escherichia coli(B) Staphylococcus aureus(C) Arthrobacter luteus(D) Haemophilus influenza
30. Describe the steps involved in the cloning of a DNA (gene of interest) into a suitable vector to form the recombinant DNA?Class on 13022023
31. Next >>>>>>>>>>>>>>Restriction MappingLigationDNA Modifying EnzymesAdditional Learning:Assigning the function of a geneCloning Strategy (OVERVIEW)Escherichia coli strainsMatings (Conjugation)Transformation
32. Experiment>>>> : Identifying Restriction Sites on a given DNA or Gene