A restriction enzyme (or Restriction endonucleases ) is an enzyme that can recognize a characteristic sequence of nucleotides within a DNA molecule and cut the DNA at that particular point called a restriction site or target or at a site not far from This, depending on the enzyme. Restriction sites have between 4 and 12 base pairs, with which they are recognized.
The mechanism of DNA cutting is done through the breaking of 2 phosphodiester bonds in the double strand, giving rise to two ends of DNA, which can be blunt (when the broken bonds coincide) or Cohesive / staggered. The latter have a tendency to rejoin spontaneously since the ends can be joined to other coincident ends that may be in the vicinity (Watson & Crick pairing).
DNA fragments obtained in this way can be linked by other enzymes called ligases . We know the vector DNA , which would be the one that is capable of replicating independently of the DNA of the host cell in which it grows. Within this group of vectors are Plasmid circular DNA molecules found in bacteria.
Restriction enzymes that despite being different and coming from different species, have the same recognition sequence and leave the same cohesive end, but do not cut at the same site, are called isoschizomers . For example, there are the Asp 718 and Kpn1 isosquizers.
The 1978 Nobel Prize in Medicine was awarded to the Microbiologists Werner Arber , Daniel Nathans and Hamilton Smith for the discovery of Restriction Endonucleases, leading to the development of recombinant DNA technology . The first practical use of his work was the manipulation of E. coli bacteria to produce human insulin for diabetics .
One of the fields in which the use of Restriction Enzymes has had greater implication has been the diagnosis of genetic diseases related to changes in the DNA sequence, whether they are point mutations , insertions or deletions of pieces. If these occur at a site of recognition of the Enzyme of Restriction, when produced they will eliminate or add new cutting sites, so when applying this enzyme to the gene of a healthy person and a sick person, different amounts of pieces should be observed for each case in an electrophoresis .
Types of Restriction Enzymes
There are, in general, 3 restriction enzyme systems:
Type 1 : A single enzyme (with 3 subunits) has restriction (short) and modification (methyla) activity. Recognizing the specific DNA sequence randomly cuts at sites other than the recognition site, either upstream or downstream. The cut leaves cohesive ends. They need ATP to move in the DNA, from the recognition site to the cutting site (about 1000 base pairs approx.), In addition to SAM (S-adenosyl-methionine) and Mg ++ as cofactors.
Type 2 : They only have restriction activity. Other enzymes carry out methylation. The cut is made at the recognition site, or close to it, so the cut is resistant and predictable. Due to this characteristic, they are widely used for gene cloning, since when cutting at specific sites, known sequences can be recovered. They only require Mg ++ as cofactor, they do not need ATP.
Type 3 : An oligomeric enzyme that performs all enzymatic activities is used. They have restriction and modification function. They cut 25 to 27 base pairs away from the recognition site, leaving cohesive ends. They require two recognition sequences in opposite orientation on the same DNA chain. They need ATP, Mg ++ and SAM (S-adenosyl-methionine) as cofactors.
There are other restriction systems currently discovered, such as the E. coli type 4 system (Eco 571) consisting of a single enzyme that cuts only methylated DNA in a specific sequence, and also methylates.
The name of each restriction enzyme is assigned according to its bacterial origin. The nomenclature used to name these enzymes consists of:
1st . Three letters that correspond to the scientific name of the microorganism (eg Escherichia coli (Eco); Haemophilus influenzae (Hin))
2nd . The strain or strain if any (eg EcoR, isolated from E. coli strain RY13)
3rd . In Roman numerals, a number to distinguish if there is more than one isolated endonuclease of that same species. Do not confuse with the type of restriction enzyme.
4th . All should carry a restriction R or an M methylase in front of the enzyme’s function, but is generally omitted.
In this way, the name of the restriction enzyme EcoRI would be constructed as follows:
|AND||Escherichia||Gender of bacteria|
|R||RY13||Strain of bacteria|
|I||The first enzyme identified||Order of identification of the enzyme in the bacteria|
Likewise, the enzymes HaeII and HaeIII come from Haemophilus aegyptius, MboI and MboII from Moraxella bovis, etc.
Examples of restriction enzymes
In this table some restriction enzymes are presented, with their respective bacterial origin and recognition site.
|Enzyme||Bacterial Origin||Recognition Site||Cut|
|Eco RI||Escherichia coli|
5 '--- G AATTC --- 3' 3 '--- CTTAA G --- 5'
|Bam HI||Bacillus amyloliquefaciens|
5 '--- G GATCC --- 3' 3 '--- CCTAG G --- 5'
|Dpn I *||Diplococcus pneumoniae|
CH 3 | 5'GATC 3'CTAG | CH 3
CH 3 | 5 '--- GA TC --- 3' 3 '--- CT AG --- 5' | CH 3
|Hin dIII||Haemophilus influenzae|
5 '--- TO AGCTT --- 3' 3 '--- TTCGA A --- 5'
|Taq I||Thermus aquaticus|
5 '--- T CGA --- 3' 3 '--- AGC T --- 5'
|Not I||Nocardia otitidis|
5 '--- GC GGCCGC --- 3' 3 '--- CGCCGG CG --- 5'
|Hin fI||Haemophilus influenzae|
5 '--- G ANTC --- 3' 3 '--- CTNA G --- 5'
|Sau 3A||Staphylococcus aureus|
5 '--- GATC --- 3' 3 '--- CTAG --- 3'
|Pov II *||Proteus vulgaris|
5 '--- CAG CTG --- 3' 3 '--- GTC GAC --- 5'
|Sma I *||Serratia marcescens|
5 '--- CCC GGG --- 3' 3 '--- GGG CCC --- 5'
|Hae III *||Haemophilus egytius|
5 '--- GG CC --- 3' 3 '--- CC GG --- 5'
|Alu I *||Arthrobacter luteus|
5 '--- AG CT --- 3' 3 '--- TC GA --- 5'
|Eco RV *||Escherichia coli|
5 '--- GAT ATC --- 3' 3 '--- CTA TAG --- 5'
|Kpn I [ 1 ]||Klebsiella pneumonia|
5 '--- GGTAC C --- 3' 3 '--- C CATGG --- 5'
|Pst I [ 1 ]||Providence stuartii|
5 '--- CTGCA G --- 3' 3 '--- G ACGTC --- 5'
|Sac I [ 1 ]||Streptomyces achromogenes|
5 '--- GAGCT C --- 3' 3 '--- C TCGAG --- 5'
|Salt I [ 1 ]||Streptomyces albue|
5 '--- G TCGAC --- 3' 3 '--- CAGCT G --- 5'
|Sph I [ 1 ]||Streptomyces phaeochromogenes|
5 '--- G CATGC --- 3' 3 '--- CGTAC G --- 5'
|Xba I [ 1 ]||Xanthomonas badrii|
5 '--- T CTAGA --- 3' 3 '--- AGATC T --- 5'
|* = DNA is left with blunt ends|