The cutting of DNA at specific boundaries
Used by restriction enzymes to locate specific sequences of DNA on which to bind and subsequently cleave
The DNA sequence to which restriction enzymes bind
The DNA sequence that is cleaved by the restriction enzyme
A Restriction Enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites.[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.
Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially[7] and are routinely used for DNA modification and manipulation in laboratories.[8][9][10]
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For the first isolation of a restriction enzyme, HindII, in 1970,[11][12] and the subsequent discovery and characterization of numerous restriction endonucleases,[13] the 1978 Nobel Prize for Physiology or Medicine was awarded to Daniel Nathans, Werner Arber, and Hamilton O. Smith.[14] Their discovery led to the development of recombinant DNA technology that allowed, for example, the large scale production of human insulin for diabetics using E. coli bacteria.[15]
Restriction enzymes recognize a specific sequence of nucleotides[2] and produce a double-stranded cut in the DNA. While recognition sequences vary between 4 and 8 nucleotides, many of them are palindromic, which correspond to nitrogenous base sequences that read the same backwards and forwards.[16] In theory, there are two types of palindromic sequences that can be possible in DNA. The mirror-like palindrome is similar to those found in ordinary text, in which a sequence reads the same forward and backwards on a single strand of DNA strand, as in GTAATG. The inverted repeat palindrome is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (i.e., of double-stranded DNA), as in GTATAC (GTATAC being complementary to CATATG).[17] Inverted repeat palindromes are more common and have greater biological importance than mirror-like palindromes.
EcoRI digestion produces "sticky" ends,
whereas 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.[18]
Different restriction enzymes that recognize the same sequence are known as neoschizomers. These often cleave in different locales of the sequence. Different enzymes that recognize and cleave in the same location are known as isoschizomers.
Restriction endonucleases are categorized into three or four 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.[19][20][21] There are four classes of restriction endonucleases: types I, II,III and IV. All types of enzymes recognise specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates. They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements,[22][23] as summarised below:
Type I restriction enzymes were the first to be identified and were first identified in two different strains (K-12 and B) of E. coli.[24] These enzymes cut at a site that differs, and is a random distance (at least 1000 bp) away, from their recognition site. Cleavage at these random sites follows a process of DNA translocation, which shows that these enzymes are also molecular motors. The recognition site is asymmetrical and is composed of two specific portions—one containing 3–4 nucleotides, and another containing 4–5 nucleotides—separated by a non-specific spacer of about 6–8 nucleotides. These enzymes are multifunctional and are capable of both restriction and modification activities, depending upon the methylation status of the target DNA. The cofactors S-Adenosyl methionine (AdoMet), hydrolyzed adenosine triphosphate (ATP), and magnesium (Mg2+) ions, are required for their full 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 the recognition (DNA-binding) site in addition to both restriction (DNA cleavage) and modification (DNA methyltransferase) activity.[19][24]
Typical type II restriction enzymes differ from type I restriction enzymes in several ways. They are a dimer of only one type of 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.[16] 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.[16] These subgroups are defined using a letter suffix.
Type IIB restriction enzymes (e.g. BcgI and BplI) are multimers, containing more than one subunit.[16] 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.[16] 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.[16] Type IIG restriction endonucleases (Eco57I) do have a single subunit, like classical Type II restriction enzymes, but require the cofactor AdoMet to be active.[16] Type IIM restriction endonucleases, such as DpnI, are able to recognize and cut methylated DNA.[16] Type IIS restriction endonucleases (e.g. FokI) cleave DNA at a defined distance from their non-palindromic asymmetric recognition sites.[16] 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.[16]
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.[26] These enzymes contain more than one subunit and require AdoMet and ATP cofactors for their roles in DNA methylation and restriction, respectively.[27] They are components of prokaryotic DNA restriction-modification mechanisms that protect the organism against invading foreign DNA. Type III enzymes are hetero-oligomeric, multifunctional proteins composed of two subunits, Res and Mod. The Mod subunit recognises the DNA sequence specific for the system and is a modification methyltransferase; as such it is functionally equivalent to the M and S subunits of type I restriction endonuclease. Res is required for restriction, although it has no enzymatic activity on its own. Type III enzymes recognise short 5-6 bp long asymmetric DNA sequences and cleave 25-27 bp downstream to leave short, single-stranded 5' protrusions. They require the presence of two inversely oriented unmethylated recognition sites for restriction to occur. These enzymes methylate only one strand of the DNA, at the N-6 position of adenosyl residues, so newly replicated DNA will have only one strand methylated, which is sufficient to protect against restriction. Type III enzymes belong to the beta-subfamily of N6 adenine methyltransferases, containing the nine motifs that characterise this family, including motif I, the AdoMet binding pocket (FXGXG), and motif IV, the catalytic region (S/D/N (PP) Y/F).[28][29]
Artificial restriction enzymes can be generated by fusing a natural or engineered DNA binding domain to a nuclease domain (often the cleavage domain of the type IIS restriction enzyme FokI[30]). Such artificial restriction enzymes can target large DNA sites (up to 36 bp) and can be engineered to bind to desired DNA sequences.[31] Zinc finger nucleases are the most commonly used artificial restriction enzymes and are generally used in genetic engineering applications,[32][33][34][35] but can also be used for more standard gene cloning applications.[36] Other artificial restriction enzymes are based on the DNA binding domain of TAL effectors.[37][38]
| Derivation of the EcoRI name | ||
|---|---|---|
| Abbreviation | Meaning | Description |
| E | Escherichia | genus |
| co | coli | species |
| R | RY13 | strain |
| I | First identified | order of identification in the bacterium |
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.[39][40] For example, the name of the EcoRI restriction enzyme was derived as shown in the box.
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 polylinker 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.[41][42]
Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing single base changes in DNA known as single nucleotide polymorphisms (SNPs).[43][44] 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).[45]
Examples of restriction enzymes include:[46]
| Enzyme | Source | Recognition Sequence | Cut |
|---|---|---|---|
| EcoRI | Escherichia coli |
5'GAATTC 3'CTTAAG |
5'---G AATTC---3' 3'---CTTAA G---5' |
| EcoRII | Escherichia coli |
5'CCWGG 3'GGWCC |
5'--- CCWGG---3' 3'---GGWCC ---5' |
| BamHI | Bacillus amyloliquefaciens |
5'GGATCC 3'CCTAGG |
5'---G GATCC---3' 3'---CCTAG G---5' |
| HindIII | Haemophilus influenzae |
5'AAGCTT 3'TTCGAA |
5'---A AGCTT---3' 3'---TTCGA A---5' |
| TaqI | Thermus aquaticus |
5'TCGA 3'AGCT |
5'---T CGA---3' 3'---AGC T---5' |
| NotI | Nocardia otitidis |
5'GCGGCCGC 3'CGCCGGCG |
5'---GC GGCCGC---3' 3'---CGCCGG CG---5' |
| HinfI | Haemophilus influenzae |
5'GANTCA 3'CTNAGT |
5'---G ANTC---3' 3'---CTNA G---5' |
| Sau3A | Staphylococcus aureus |
5'GATC 3'CTAG |
5'--- GATC---3' 3'---CTAG ---5' |
| PovII* | Proteus vulgaris |
5'CAGCTG 3'GTCGAC |
5'---CAG CTG---3' 3'---GTC GAC---5' |
| SmaI* | Serratia marcescens |
5'CCCGGG 3'GGGCCC |
5'---CCC GGG---3' 3'---GGG CCC---5' |
| HaeIII* | Haemophilus aegyptius |
5'GGCC 3'CCGG |
5'---GG CC---3' 3'---CC GG---5' |
| HgaI[47] | Haemophilus gallinarum |
5'GACGC 3'CTGCG |
5'---NN NN---3' 3'---NN NN---5' |
| AluI* | Arthrobacter luteus |
5'AGCT 3'TCGA |
5'---AG CT---3' 3'---TC GA---5' |
| EcoRV* | Escherichia coli |
5'GATATC 3'CTATAG |
5'---GAT ATC---3' 3'---CTA TAG---5' |
| EcoP15I | Escherichia coli |
5'CAGCAGN25NN 3'GTCGTCN25NN |
5'---CAGCAGN25NN ---3' 3'---GTCGTCN25 NN---5' |
| KpnI[48] | Klebsiella pneumoniae |
5'GGTACC 3'CCATGG |
5'---GGTAC C---3' 3'---C CATGG---5' |
| PstI[48] | Providencia stuartii |
5'CTGCAG 3'GACGTC |
5'---CTGCA G---3' 3'---G ACGTC---5' |
| SacI[48] | Streptomyces achromogenes |
5'GAGCTC 3'CTCGAG |
5'---GAGCT C---3' 3'---C TCGAG---5' |
| SalI[48] | Streptomyces albus |
5'GTCGAC 3'CAGCTG |
5'---G TCGAC---3' 3'---CAGCT G---5' |
| ScaI[48] | Streptomyces caespitosus |
5'AGTACT 3'TCATGA |
5'---AGT ACT---3' 3'---TCA TGA---5' |
| SpeI | Sphaerotilus natans |
5'ACTAGT 3'TGATCA |
5'---A CTAGT---3' 3'---TGATC A---5' |
| SphI[48] | Streptomyces phaeochromogenes |
5'GCATGC 3'CGTACG |
5'---GCATG C---3' 3'---C GTACG---5' |
| StuI[49][50] | Streptomyces tubercidicus |
5'AGGCCT 3'TCCGGA |
5'---AGG CCT---3' 3'---TCC GGA---5' |
| XbaI[48] | Xanthomonas badrii |
5'TCTAGA 3'AGATCT |
5'---T CTAGA---3' 3'---AGATC T---5' |
Key:
* = blunt ends
N = C or G or T or A
W = A or T
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