With the advancement in biotechnology, restriction enzyme has become an indispensable tool for recombinant DNA technology.
A restriction enzyme also known as molecular scissors is a site-specific endonuclease encoded by bacteria and archaea. This article accounts for detailed facts about different restriction enzyme examples.
Different types of restriction enzymes:
Restriction enzyme examples and its recognition sites are listed below.
Source– Escherichia coli
Recognition sequence – 5’-GAATTC-3′ 3’-CTTAAG-5′ ; sticky ends
Source –Escherichia coli
Recognition sequence – 5’-CCWGG-3′ 3’-GGWCC-5′; sticky ends
Recognition sequence – 5’-GGATCC-3′ 3’-CCTAGG-5′: sticky ends
Source –Thermus aquaticus
Recognition sequence- 5’ TCGA-3′ 3’-AGCT-5′
Source – Haemophilus influenzae
Recognition sequence – 5’ AAGCTT-3′ 3’-TTCGAA -5′
Source – Staphylococcus aureus
Recognition sequence – 5′ GATC-3′ 3′-CTAG-5′;
Source –Nocardia otitidis
Recognition sequence – 5′-GCGGCCGC-3′ 3′-CGCCGGCG-5′
Source –Proteus vulgaris
Recognition sequence – 5′-CAGCTG-3′ 3′-GTCGAC-5′
Source – Haemophilus aegyptius
Recognition sequence- 5′ GGCC-3′ 3′-CCGG-5′
Source – Arthrobacter luteus
Recognition sequence- 5’-AGCT-3′ 3’-TCGA-5′
Source – Escherichia coli
Recognition sequence- 5′-GATATC- 3′ 3′-CTATAG- 5′
Recognition sequence – 5′ -GTCGAS-3′ 3′-CAGCTG -5′
Recognition sequence- 5′-AGTACT-3′ 3′-TCATGA-5′
Source Serratia marcescens
Recognition sequence – 5′-CCCGGG-3′ 3′-GGGCCC-5′
Source Haemophilus influenzae
Recognition sequence- 5′-GANTC-3′ 3′-CTNAG-5′
Source Haemophilus aegyptius
Recognition sequence- 5′-GGCC-3′ 3′- CCGG-5′
Source Haemophilus gallinarum
Recognition sequence- 5’GACGC-3′ 3′-CTGCG-5′
Source Escherichia coli
Recognition sequence- 5′-CAGCAGN25NN-3′ 3′-GTCGTCN25NN-5′
Source – Klebsiella pneumoniae
Recognition sequence– 5’GGTACC-3′ 3′-CCATGG-5′
Source – Providencia stuartii
Recognition sequence– 5′-CTGCAG-3′ 3′-GACGTC-5′
Source – Streptomyces achromogenes
Recognition sequence- 5′-GAGCTC-3′ 3′-CTCGAG-5′
Sorurce –Sphaerotilus natans
Recognition sequence- 5′-ACTAGT-3′ 3′-TGATCA-5′
Source – Streptomyces phaeochromogenes
Recognition sequence- 5′-GCATGC-3′ 3′-CGTACG-5′
Source- Streptomyces tubercidicus
Recognition sequence- 5′-AGGCCT-3′ 3′-TCCGGA-5′
Source –Xanthomonas badrii
Recognition sequence – 5′-TCTAGA-3′ 3′-AGATCT-5′
Restriction enzymes cut the DNA strands in two different ways.
Some restriction endonuclease cut the DNA at the same point. Such straight-cut within the recognition site creates blunt ends with no over-hanging ends. These ends are often ligated by DNA ligase enzymes. Examples: PvuII, Haelll, Alul.
Contrastingly, the second class of restriction endonuclease undergoes staggered cut resulting in complementary single-stranded over-hanging ends. Such ends are called sticky or cohesive ends. Examples EcoR1, BamH1, Taq1. These are frequently used for cloning purposes in biotechnology.
Types of restriction enzymes
Restriction enzymes can be classified into four groups depending upon the composition, type of cofactors requirements, the target site, and cleavage position.
Type I –
This type of restriction enzyme are multifunctional proteins that cleave only one DNA strand at random as well as distant sites and also performs methylase activities. The target sequence is about 15 bp in length and cleaves non-specifically away from the recognition site. For catalytic activity, it requires Mg2+, ATP, and S-adenosyl – L- methionine. Examples – EcoK, EcoB.
This group comprises most orthodox restriction enzymes which are used in recombinant DNA technology. These enzymes are the most stable endonucleases which cleave DNA at specific sites. Thus, these generate desirable fragments of DNA. The recognition sequences are palindromic in nature of 4-8 bp and for catalytic activity, only Mg2+ ions are required. These enzymes can perform the nucleolytic activity only. Examples – Hinfl, EcoRI, PvuII, Alul, Haelll.
This group is an intermediate type between type I and type II. The length of the recognition sequence is 24-26 bp. They require both Mg2+ and ATP for their activity and cleave DNA sequences in close vicinity of target sites. Eg. Hinf III, EcoP1.
The target DNA for this group is different from the rest of the types. It recognizes modified DNA sequences such as methylated, hydroxy-methylated, and also glycosylated bases. Eg., McrBC.
Restriction enzyme nomenclature
Restriction enzymes have unique nomenclature. Each enzyme is named after the bacterium from where it is isolated. The name contains the genus, species, and the strain of the bacterium.
The nomenclature of restriction enzymes follows a pattern
1. The first capital letter is the name of the genus from which bacterium is discovered.
2. The first two letters of the species name are written after the first initial.
3. Next is the strain identified which is written in subscript.
4. The number of enzymes produced by the bacterium.
5. Generally all restriction enzymes are prefixed with the general symbol R. This is used to distinguish from the methylases that are obtained from the same strains.
Example: EcoRI name
E Escherichia genus
co coli species
R RY13 strain
I First identified order of identification in the bacterium
What is a restriction enzyme?
The term restriction enzyme was derived from the studies of lambda bacteriophages where it was observed that these bacterial protein enzymes cleave the phage DNA and thus, restrict the activity. The own target sites of the bacterial cell are high methylated i.e., the addition of methyl groups to the adenosine and cytosine bases within the recognition sites. This methylation protects from cleavage.
A restriction enzyme is an endonuclease that enables site-specific cleavage of DNA sequence. These sites are called restriction sites or recognition sequences or target sites. These are usually synthesized by bacteria for defense mechanisms against invading bacteriophages. The mechanism comprising methylation along with restriction enzyme activity constitutes the restriction-modification system.
DNA strand contains two strands. 5’ end-3’ end depicts the forward strand while the 3’ end – 5’ end is denoted as a reverse strand. At first, restriction enzymes recognize the specific DNA sequences and then make two incisions on each strand of DNA sequence. This specific sequence is called recognition sites. The sequences of recognition sites are palindrome sequences which reads the same on forward and reverse strands when read in the same orientation. The recognition sequences usually contain 4-8 nucleotides, mostly palindromic in nature.
Restriction enzyme structure
The most convenient restriction endonuclease enzymes belong to the type II enzyme.
The recognition sites are typically a short palindromic sequence of 4-8 bp and catalytic activity requires Mg2+ ions. It consists of two homodimers each 30kDa molecular mass, that recognize the palindromic sequences. The structural core of these enzymes consists of four β-strands and one α-helix. For this nucleolytic activity, it doesn’t require ATP hydrolysis.
As restriction enzymes are target-specific, 15-20 hydrogen bonds are formed between the dimers and bases of the target site. In addition to hydrogen bonds, Van der Waals interaction also takes place. Due to these interactions, the enzyme undergoes conformational change leading to the activation of the catalytic center of the restriction enzyme. The catalytic center contains two carboxylates which are necessary for the binding of cofactor Mg2+. The resultants of the catalysis are DNA fragments with 5’-P and 3’-OH.
Restriction enzyme is a site- specific endonuclease that cleaves DNA strands at specific recognition sites. These are synthesized by bacteria as a part of their defense mechanism. For its activity some requires magnesium ions while others required ATP and S-adenosyl – L- methionine. Due to its nucleolytic activity these enzymes are extensively used in recombinant DNA technology.