Restriction enzyme

A restriction enzyme (or restriction endonuclease) is one that can recognize a characteristic sequence of nucleotides within a DNA molecule and cut the DNA at that point specifically, called the restriction site or target, or at a site not far from it. Restriction sites have between four and six base pairs, with which they are recognized.

The mechanism of DNA cutting is carried out through the breaking of two phosphodiester bonds in the double strand, which gives rise to two ends of DNA. These can be blunt (when the broken links coincide) or cohesive/staggered. The latter have a tendency to join again spontaneously, since the ends can join other coincident ends that may be in the vicinity (Watson & Crick mating).

Cutting EcoRI leaving cohesive ends.

Restriction SmaI'm leaving extremes.

The DNA fragments obtained in this way can be joined together by other enzymes called ligases.

Restriction enzymes that, despite being different and coming from different species, have the same recognition sequence and leave the same sticky end, but do not cut in the same place, are called isoschizomers. For example, there are the isoschizomers Asp718 and KpnI.

The 1978 Nobel Prize in Medicine was awarded to microbiologists Werner Arber, Daniel Nathans and Hamilton Smith for the discovery of restriction endonucleases which led to the development of recombinant DNA technology. The first practical use of his work was the manipulation of the bacterium E. coli to produce human insulin for diabetics.

One of the fields in which restriction enzymes have had the greatest implication has been the diagnosis of genetic diseases related to changes in the DNA sequence, whether point mutations, insertions or deletions of fragments. If these occur at a restriction enzyme recognition site, when produced they will remove or add new cleavage sites. When applying this enzyme to the gene of a healthy person and a sick person, different amounts of fragments should be observed for each case in an electrophoresis.

Restriction-modification (R-M) systems

The restriction-modification (M-R) system is used in vivo by bacteria to protect themselves from exogenous (usually viral) DNA attacks that enter the bacteria, eliminating foreign sequences from the bacteria's genome. Own DNA is not deleted by the restriction enzymes of the organism that produces it, since it has previously been modified by methylation through the action of a methyltransferase, an enzyme that transfers methyl groups from S-adenosyl-methionine (SAM). to specific bases. This system was discovered in 1968, as a result of a series of studies on phage I infection in two different strains of Escherichia coli (the so-called “K12” and “B” strains). This system consists of two parts:

• Restriction: This system allows bacteria to protect themselves from exogenous DNA to avoid “promiscuity” in genetic exchanges. This gives you immunity from bacteriophages that could endanger the genetic individuality or even the life of the bacteria, which is done through cuts through endonucleases of restriction to exogenous DNAs that enter the bacteria.

• Modification: Consists in the introduction of methyl groups (CH)₃-) in certain bases within certain DNA sequences, which is catalyzed by a specific metylase.

There are several general mechanisms of action of these systems, of which type I and type II stand out:

• M-R type I: They were the first to be described. The characteristic of type I M-R systems is that modification and restriction activities are produced by a multiprotein complex, which performs the two types of activities. Some features are:

• They recognize relatively large sequences that do not present symmetries.

• The restriction activity (of the R subunits) cuts away from the reconnaissance site (about 1000 pb), and in unspecific places.

• The restriction requires ATP.

• The metilasa activity of the complex makes use of S-adenosil-methionine (SAM) as a donor of the methyl groups.

• M-R type II: Here each subunit of the dimer recognizes the same sequence, present in each of the speculative parts of the pallindrome, and performs a cut in a specific place, which obviously has its corresponding in the other chain of the other half of the pallindrome. More than one third of bacterial strains have at least one type II M-R system. Many of them are encoded from genes located in plasmids. Some features that this mechanism has are:

• The two activities are carried out by two different proteins that do not form multiprotein complexes.

• They don't use ATP. They just need Mg ions.⁺ to work. Methylase uses SAM as a meth donor.

• Each member of the modification and restriction couple recognizes the same specific DNA sequence.

• The reconnaissance site consists of 4 or 6 pbs that constitute a palinotronomic sequence.

• Methylase is usually a monomeric protein, which introduces methyl groups into an A or a G (according to the methylase in question), in both chains, within the recognition site.

• The restriction enzyme is usually a protein formed by two subunits of the same type, assembled symmetrically. They can leave cohesive extremes or end romos. Some restriction enzymes leave outstanding 5' ends, while others leave outstanding 3' ends.

Restriction enzyme model

There are, in general, 3 restriction enzyme systems:

• Model 1: A single enzyme (with 3 subunits) has restrictive activity (cut) and modification (methyl). By acknowledging the specific sequence of DNA cut randomly in different sites to the site of recognition, either upstream or downstream. The cut leaves cohesive ends. They need ATP to move in DNA, from the place of recognition to the cutting site (about 1000 pairs of bases), as well as SAM (S-adenosil-metionine) and Mg⁺ like cofactors.

• Model 2: They only have restrictive activity. Other enzymes carry out methylation. The cut is made on the recognition site, or near it, so the cut is resistant and predictable. This feature is that they are widely used for gene cloning, as cutting into specific sites can recover known sequences. They only require Mg⁺ as a cofactor, they don't need ATP.

• Model 3: An oligomeric enzyme is used that performs all enzyme activities. They have restriction and modification function. They cut from 25 to 27 base pairs away from the recognition site, leaving cohesive ends. They require two recognition sequences in opposite orientation in the same DNA chain. They need ATP, Mg⁺ and SAM (S-adenosil-methionine) as cofactors.

There are other restriction systems currently discovered, such as the type 4 system of E. coli (Eco571) consisting of a single enzyme that cuts only methylated DNA in a specific sequence, and also methylates.

Nomenclature

• 1o. Three letters corresponding to the scientific name of the microorganism (e.g. Escherichia coli (Eco); Haemophilus influenzae (Hin)); and therefore the first three letters of the name are written in italics.

• 2nd. The strain or stretch if there was (e.g. EcoRisolated from the strain "RY13" of E. coli)

• 3o. In Roman numerals, a number to distinguish if there is more than one endonuclease isolated from that same species. Do not confuse with the type of restriction enzyme.

• 4o. All should carry a restraining R or a metilasa M according to the function of the enzyme, but it is usually omitted.

In this way, the name of the restriction enzyme EcoRI would be constructed as follows:

Likewise, the enzymes HaeII and HaeIII come from Haemophilus aegyptius, MboI and MboII from Moraxella bovis, etc.

Examples

Check the main article list of restricted enzyme cutting sites.

This table presents some restriction enzymes, with their respective bacterial origin and recognition site.

5'GAATTC
3'CTTAAG

5'---G AATTC---3'
3'---CTTAA G---5'

5'GGATCC
3'CCTAGG

5'---G GATCC---3'
3'---CCTAG G---5'

5'AGATCT
3'TCTAGA

5'---A GATCT---3'
3'---TCTAG A---5'

5'GGATG
3'CCTAC

 CH3 日本語5'GATC
3'CTAG
 日本語CH3

 CH3 日本語5'---GA TC---3'
3'---CT AG---5'
 日本語CH3

5'GTPyPuAC
3'CAPuPyTG

5' ---GTPy PuAC---3'
3' ---CAPu PyTG---5'

5'AAGCTT
3'TTCGAA

5'---A AGCTT---3'
3'---TTCGA A---5'

5'TCGA
3'AGCT

5'---T CGA---3'
3'---AGC T---5'

5'GCGCCGC
3'CGCCGGCG

5'---GC GGCCGC---3'
3'---CGCCGG CG---5'

5'GANTC
3'CTNAG

5'---G ANTC---3'
3'---CTNA G---5'

5'GATC
3'CTAG

5'--- GATC---3'
3'---CTAG ---5'

5'CAGCTG
3'GTCGAC

5'---CAG CTG---3'
3'---GTC GAC---5'

5'CCCGGGG
3'GGCCC

5'---CCC GGG---3'
3'---GGG CCC---5'

5'GGCC
3'CCGG

5'---GG CC---3'
3'---CC GG---5'

5'AGCT
3'TCGA

5'---AG CT---3'
3'---TC GA---5'

5'GATATC
3'CTATAG

5'---GAT ATC---3'
3'---CTA TAG---5'

5'GGTACC
3'CCATGG

5'---GGGTAC C---3'
3'---C CATGG---5'

5'CTGCAG
3'GACGTC

5'---CTGCA G---3'
3'---G ACGTC---5'

5'GAGCTC
3'CTCGAG

5'---GAGCT C---3'
3'---C TCGAG---5'

5'GTCGAC
3'CAGCTG

5'---G TCGAC---3'
3'---CAGCT G---5'

5'GCATGC
3'CGTACG

5'---G CATGC---3'
3'---CGTAC G---5'

5'TCTAGA
3'AGATCT

5'---T CTAGA---3'
3'---AGATC T---5'

Applications in the diagnosis of genetic diseases

Thanks to restriction enzymes we are able to diagnose certain genetic diseases, either due to a variation in the genetic material (duplication, deletion, etc.) or due to a mutation on a specific basis. The procedure for said diagnosis would consist of amplifying by PCR a specific region of the chromosome where we know that this genetic change should or should not be present and adding the restriction enzyme or enzymes so that the pertinent cuts are made. After making these cuts, we will run our samples in an electrophoresis gel and we will see the differences in sizes that exist. If the mutation generates a deletion, when running in the electrophoresis we will observe a fragment smaller than what we would expect from a healthy patient, in such a way that we could diagnose it. If the mutation, on the other hand, generates a point mutation, we can take advantage of it to use an enzyme at that point and thus, if there is no such mutation, that cut will not occur and we will obtain a larger fragment in the electrophoresis gel.