Structure of the homodimeric restriction enzyme EcoRI (cyan and green cartoon diagram) bound to double stranded DNA (brown tubes) based on the X-ray crystallographic coordinates. Two catalytic manganese ions (one from each monomer) are shown as magenta spheres and are adjacent to the cleaved sites in the DNA made by the enzyme (depicted as gaps in the DNA backbone).

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA following its specific recognition of short nucleotide sequences, known as restriction sites, in the DNA.^[1][2][3] Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses.^[4][5] Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction; host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme?s activity. Collectively, these two processes form the restriction modification system.^[6] To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.

The Nobel Prize in Medicine was awarded, in 1978, to Daniel Nathans, Werner Arber, and Hamilton Smith for the discovery of restriction endonucleases.^[7] Their discovery lead to the development of recombinant DNA technology that allowed, for example, the large scale production of human insulin for diabetics using E. coli bacteria.^[8] Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially.^[9] and are routinely used for DNA modification and manipulation in laboratories.^[10][11][12]

Recognition sites

5'-GTATAC-3'

   

3'-CATATG-5'

Restriction enzymes recognize a specific sequence of nucleotides^[2] and produce a double-stranded cut in the DNA that prevents the phage from replicating. While recognition sequences vary widely, with lengths between 4 and 8 nucleotides, many of them are palindromic; that is, the sequence on one strand reads the same in the reverse direction on the complementary strand.^[13] The meaning of "palindromic" in this context is different from what one might expect from its linguistic usage: GTAATG is not a palindromic DNA sequence, but GTATAC is (GTATAC is complementary to CATATG).

EcoRI digestion produces "sticky" ends	SmaI restriction enzyme cleavage produces "blunt" ends

Recognition sequences in DNA differ for each restriction enzyme, producing differences in the length, sequence and strand orientation (5' end or the 3' end) of a sticky-end "overhang" of an enzyme restriction.^[14]

Different restriction enzymes that recognize the same sequence are known as neoschizomers. These often cleave in a different location of the sequence; however, the specific type that cleaves in the same location as the prototype is known as an isoschizomer.

Bacteria prevent their own DNA from being cut by modifying their nucleotides via DNA methylation.^[4]

Enzyme classes

Restriction endonucleases are categorized into three general groups (Types I, II and III) based on their composition and enzyme cofactor requirements, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence.^[15][16][17]

Type I

Type I restriction enzymes were the first to be identified and are characteristic of two different strains (K-12 and B) of E. coli.^[18] These enzymes cut at a site that differs, and is some distance (at least 1000 bp) away, from their recognition site. The recognition site is asymmetrical and is composed of two portions ? one containing 3-4 nucleotides, and another containing 4-5 nucleotides ? separated by a spacer of about 6-8 nucleotides. Several enzyme cofactors, including S-Adenosyl methionine (AdoMet), hydrolyzed adenosine triphosphate (ATP) and magnesium (Mg²⁺) ions, are required for their activity. Type I restriction enzymes possess three subunits called HsdR, HsdM, and HsdS; HsdR is required for restriction, HsdM is necessary for adding methyl groups to host DNA (methyltransferase activity) and HsdS is important for specificity of cut site recognition in addition to its methyltransferase activity.^[15][18]

Type II

Typical type II restriction enzymes differ from type I restriction enzymes in several ways. They are composed of only one subunit, their recognition sites are usually undivided and palindromic and 4-8 nucleotides in length, they recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their activity ? they usually require only Mg²⁺ as a cofactor.^[13] These are the most commonly available and used restriction enzymes. In the 1990s and early 2000s, new enzymes from this family were discovered that did not follow all the classical criteria of this enzyme class, and new subfamily nomenclature was developed to divide this large family into subcategories based on deviations from typical characteristics of type II enzymes.^[13] These subgroups are defined using a letter suffix.

Type IIB restriction enzymes (e.g. BcgI and BplI) are multimers, containing more than one subunit.^[13] They cleave DNA on both sides of their recognition to cut out the recognition site. They require both AdoMet and Mg²⁺ cofactors. Type IIE restriction endonucleases (e.g. NaeI) cleave DNA following interaction with two copies of their recognition sequence.^[13] One recognition site acts as the target for cleavage, while the other acts as an allosteric effector that speeds up or improves the efficiency of enzyme cleavage. Similar to type IIE enzymes, type IIF restriction endonucleases (e.g. NgoMIV) interact with two copies of their recognition sequence but cleave both sequences at the same time.^[13] Type IIG restriction endonucleases (Eco57I) do have a single subunit, like classical Type II restriction enzymes, but require the cofactor AdoMet to be active.^[13] Type IIM restriction endonucleases, such as DpnI, are able to recognize and cut methylated DNA.^[13] Type IIS restriction endonucleases (e.g. FokI) cleave DNA at a defined distance from their non-palindromic asymmetric recognition sites.^[13] These enzymes may function as dimers. Similarly, Type IIT restriction enzymes (e.g., Bpu10I and BslI) are composed of two different subunits. Some recognize palindromic sequences while others have asymmetric recognition sites.^[13]

Type III

Type III restriction enzymes (e.g. EcoP15) recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20-30 base pairs after the recognition site.^[19] These enzymes contain more than one subunit and require AdoMet and ATP cofactors for their roles in DNA methylation and restriction, respectively.^[20]

Nomenclature

Since their discovery in the 1970s, more than 100 different restriction enzymes have been identified in different bacteria. Each enzyme is named after the bacterium from which it was isolated using a naming system based on bacterial genus, species and strain.^[21][22] For example, the name of the EcoRI restriction enzyme was derived as shown in the box.

Restriction enzymes as tools

See the main article on restriction digests.

Isolated restriction enzymes are used to manipulate DNA for different scientific applications.

• They are used to assist insertion of genes into plasmid vectors during gene cloning and protein expression experiments. For optimal use, plasmids that are commonly used for gene cloning are modified to include a short linker sequence (called the multiple cloning site, or MCS) rich in restriction enzyme recognition sequences. This allows flexibility when inserting gene fragments into the plasmid vector; restriction sites contained naturally within genes influence the choice of endonuclease for digesting the DNA since it is necessary to avoid restriction of wanted DNA while intentionally cutting the ends of the DNA. To clone a gene fragment into a vector, both plasmid DNA and gene insert are typically cut with the same restriction enzymes, and then glued together with the assistance of an enzyme known as a DNA ligase.^[23][24]

• They can be used to distinguish gene alleles by specifically recognizing single base changes in DNA known as single nucleotide polymorphisms (SNPs).^[25][26] This is only possible if a SNP alters the restriction site present in the allele. In this method, the restriction enzyme can be used to genotype a DNA sample without the need for expensive gene sequencing. The sample is first digested with the restriction enzyme to generate DNA fragments, and then the different sized fragments separated by gel electrophoresis. In general, alleles with correct restriction sites will generate two visible bands of DNA on the gel, and those with altered restriction sites will not be cut and will generate only a single band. The number of bands reveals the sample subject's genotype, an example of restriction mapping.

• In a similar manner, restriction enzymes are used to digest genomic DNA for gene analysis by Southern Blot. This technique allows researchers to identify how many copies (or paralogues) of a gene are present in the genome of one individual, or how many gene mutations (polymorphisms) have occurred within a population. The latter example is called Restriction Fragment Length Polymorphism (RFLP).^[27]

Examples

Examples of restriction enzymes include:^[28]

5'GAATTC
3'CTTAAG

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

5'CCWGG
3'GGWCC

5'---     CCWGG---3'
3'---GGWCC     ---5'

5'GGATCC
3'CCTAGG

5'---G     GATCC---3'
3'---CCTAG     G---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'GCGGCCGC
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'CCCGGG
3'GGGCCC

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'---GGTAC  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'AGTACT
3'TCATGA

5'---AGT  ACT---3'
3'---TCA  TGA---5'

5'GCATGC
3'CGTACG

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

5'AGGCCT
3'TCCGGA

5'---AGG  CCT---3'
3'---TCC  GGA---5'

5'TCTAGA
3'AGATCT

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

See also

• Star activity
• molecular weight size marker

References

External links

General:

Databases:

Software:

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