Restriction endonuclease and it’s classification, properties, uses in molecular biology

Restriction endonuclease

Restriction enzyme, also called restriction endonuclease, a protein produced by bacteria that cleaves DNA at specific sites along the molecule. In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason they are indispensable tools of recombinant DNA technology (genetic engineering).


Restriction enzymes are traditionally classified into four types on the basis of subunit composition, cleavage position, sequence specificity and cofactor requirements. However, amino acid sequencing has uncovered extraordinary variety among restriction enzymes and revealed that at the molecular level, there are many more than four different types.

Type I enzymes are complex, multi-subunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences. Originally thought to be rare, we now know from the analysis of sequenced genomes that they are common. Type I enzymes are of considerable biochemical interest, but they have little practical value since they do not produce discrete restriction fragments or distinct gel-banding patterns.

Type II enzymes cut DNA at defined positions close to or within their recognition sequences. They produce discrete restriction fragments and distinct gel banding patterns, and they are the only class used in the laboratory for routine DNA analysis and gene cloning. Rather than forming a single family of related proteins, Type II enzymes are a collection of unrelated proteins of many different sorts. Type II enzymes frequently differ so completely in amino acid sequence from one another, and indeed from every other known protein, that they exemplify the class of rapidly evolving proteins that are often indicative of involvement in host-parasite interactions.

The most common Type II enzymes are those like HhaI (NEB #R0139), HindIII (NEB #R0104), and NotI (NEB #R0189), that cleave DNA within their recognition sequences. Enzymes of this kind are the principal ones available commercially. Most recognize DNA sequences that are symmetric, because they bind to DNA as homodimers, but a few, (e.g., BbvCI (NEB #R0601): CCTCAGC) recognize asymmetric DNA sequences, because they bind as heterodimers. Some enzymes recognize continuous sequences (e.g., EcoRI (NEB #R0101): GAATTC) in which the two half-sites of the recognition sequence are adjacent, while others recognize discontinuous sequences (e.g., BglI (NEB#R0144): GCCNNNNNGGC) in which the half-sites are separated. Cleavage leaves a 3´-hydroxyl on one side of each cut and a 5´-phosphate on the other. They require only magnesium for activity and the corresponding modification enzymes require only S-adenosylmethionine. They tend to be small, with subunits in the 200-350 amino acid range.

The next most common Type II enzymes, usually referred to as ‘Type IIS” are those like FokI (NEB #R0109) and AlwI (NEB #R0513) that cleave outside of their recognition sequence to one side. These enzymes are intermediate in size, 400-650 amino acids in length, and they recognize sequences that are continuous and asymmetric. They comprise two distinct domains, one for DNA binding, the other for DNA cleavage. They are thought to bind to DNA as monomers for the most part, but to cleave DNA cooperatively, through dimerization of the cleavage domains of adjacent enzyme molecules. For this reason, some Type IIS enzymes are much more active on DNA molecules that contain multiple recognition sites.

Type IIG restriction enzymes, the third major kind of Type II enzyme, are large, combination restriction-and-modification enzymes, 850-1250 amino acids in length, in which the two enzymatic activities reside in the same protein chain. These enzymes cleave outside of their recognition sequences and can be classified as those that recognize continuous sequences (e.g., AcuI (NEB #R0641): CTGAAG) and cleave on just one side; and those that recognize discontinuous sequences (e.g., BcgI (NEB #R0545): CGANNNNNNTGC) and cleave on both sides releasing a small fragment containing the recognition sequence. The amino acid sequences of these enzymes are varied, but their organization is consistent. They comprise an N-terminal DNA-cleavage domain joined to a DNA-modification domain and one or two DNA sequence-specificity domains forming the C-terminus or present as a separate subunit. When these enzymes bind to their substrates, they switch into either restriction mode to cleave the DNA, or modification mode to methylate it.

The type II enzymes do not require ATP for their activity and they cleave close to or within the recognition site. These enzymes are the most extensively used enzymes for gene analysis and cloning work, and are classified into several subtypes:7

Subtype Characteristic Features Example Enzymes
Type II
  • Cleavage within or adjacent to the recognition site
  • Palindromic recognition site
  • Homodimeric enzyme
EcoR I (R6265)
Type IIS
  • Cleavage at a defined distance from the recognition site
  • Asymmetric recognition site
Fok I
Type IIE
  • Interact with two recognition sites
  • One serves as the allosteric effector and the other site is for cleavage
Nae I
Type IIF
  • Interact with two recognition sites
  • Both are required for cleavage
  • Homotetrameric enzyme
Type IIT
  • Contain different subunits with restriction and modification activities
Bpu10 I
Type IIG
  • Comprised of a single polypeptide chain with restriction and modification activity
Type IIB
  • Cleave both sides of the recognition site
Bcg I
Type IIM
  • Recognize methylated sites
Dpn I (R8381)

Type III enzymes are also large combination restriction-and-modification enzymes. They cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage; they rarely give complete digests.

Type IV enzymes recognize modified, typically methylated DNA and are exemplified by the McrBC and Mrr systems of E. coli.

Essential Properties of Restriction Enzymes

DNA restriction and modification systems are common mechanisms by which bacteria protect their DNA from contamination by invading or foreign DNA. 
Many bacteria produce a restriction enyzme that cleaves foreign DNA at a specific sequence. 
For the bacteria to survive the presence of these enzymes, an accompanying DNA modification system (such as adenine methylation and cytosine methylation)

Type II restriction/modification systems consist of two separate proteins: 
    1) a restriction endonuclease and 
    2) a separate methylase. 
Note that Type I and III restriction/modification systems have both activities included within one protein.

Restriction enzymes bind to and cleave double-stranded DNA at specific sites. 
Different restriction sites recognize different sequences.

Most type II restriction enzymes recognize symmetric sequences that are 4, 5 or 6 base pairs in length. 
A small minority of restriction enzymes recognize larger sequences. 
Some recognize somewhat non-symmetric sequences. 
The site of cleavage within the recognition sequence can produce 
    1) blunt ends, 
    2) overhanging 3 prime ends or 
    3) overhanging 5 prime ends. 
The protruding single-strands can be united with similar ends to readily produce recombinant molecules.

Isoschizomers are enzymes from different sources that target the same target sequence. 
Depending upon how the cleavage sites compare, the enzymes may or may leave the same ends (compatible ends). 
Often enzymes with a hexanucleotide recognition sequence (6-cutters) will leave a 4 nucleotide overhang. 
Other enzymes that recognize a different hexanucleotide sequence that share the same central tetranucleotide motif may leave the same ends (compatible ends).

For example: SalI (GTCGAC) and XhoI (CTCGAG) leave compatible ends that once ligated, result in a site (GTCGAG) that is not cleaved by either enzyme.

Other  areas of concern when using Restriction enzymes 
Double digestion conditions 
Heat inactivation 
Activity near DNA fragment ends 
Star activity 
Sensitivity to methylation

Restriction mapping 
A segment of DNA can be characterized by breaking the molecule at defined sites. 
The distance between the sites can calculated. 
Map can be constructed. 


The ability of restriction endonucleases to cleave DNA at specific recognition sites has enabled extensive use of these enzymes as essential tools in several molecular biology techniques. Some of the major applications are explained below:

  1. Molecular cloning: A popular application of restriction enzymes has been in the generation of recombinant DNA molecules. The process involves the cutting of the donor DNA (usually a plasmid) and the vector DNA (usually a gene from another organism) by a restriction enzyme to yield compatible ends. These ends could be either ‘blunt’ or ‘sticky’. The two cleaved DNAs are joined together using an enzyme called DNA ligase to generate a recombinant DNA molecule. This recombinant DNA can, then, be introduced into a host organism for replication. For more details, refer Restriction Enzyme Cloning Manual.
  2. DNA mapping, also known as restriction mapping, involves the use of restriction endonucleases to obtain structural information of the DNA fragment or genome. Mapping involves determination of the order of the restriction enzyme sites in the genome. The DNA of interest, whose structure is to be determined, is cleaved with a series of restriction endonucleases to produce DNA fragments varying in size. These fragments are separated on an agarose gel to determine the structure of the DNA of interest.
    Based on the known restriction enzyme sites of a specific DNA fragment, restriction endonucleases can be used to verify the identity of that DNA fragment.
  3. Restriction landmark genomic scanning is a genome analysis method that utilizes a combination of restriction enzymes to visualize differences in methylation levels across the genome of a given organism. It is a useful technique to identify deviations from normal in any DNA. It is very effective in detecting hyper/hypomethylation in tumors, deletions or amplifications of genes, or changes in gene expression throughout the development of an organism.
  4. Gene sequencing: A large DNA molecule can be sequenced by digesting it with restriction enzymes and processing the resulting fragments through a DNA sequencer.
  5. Restriction fragment length polymorphism (RFLP) involves the digestion of a DNA sample using restriction enzymes, separating these fragments based on length by gel electrophoresis and transferring them onto a membrane. These fragments are then bound to a radioactive or fluorescent labeled probe targeting specific sequences that are bracketed by restriction enzyme sites. An RFLP occurs when the resulting fragment lengths vary between individuals. Each individual has a unique pattern called the ‘biological bar code’. This technique was the first DNA profiling technique used in gene mapping, localization of genes for genetic disorders, determination of risk for disease, and paternity testing.
  6. Pulse field gel electrophoresis involves the separation of large DNA fragments, mainly the fragments resulting from digesting a bacterial genome with a rare-cutting restriction enzyme. The unique pattern produced is used to distinguish different strains of bacteria. It may be useful to identify a particular strain as the cause of a widespread disease.
  7. Serial analysis of gene expression (SAGE) is a technique that involves the quantitative and simultaneous analysis of a large number of transcripts in the form of small tags. Restriction enzymes are used as an anchoring enzyme and a tagging enzyme in this technique.
  8. Restriction enzyme-mediated integration (REMI) involves the use of restriction enzymes to produce compatible cohesive ends in the genome for the insertion of a mixture of plasmid DNA that has been linearized with a restriction enzyme. The plasmid DNA is transformed into the host cell along with a restriction enzyme that facilitates the integration of the DNA into cognate restriction sites in the chromosomes. This technique is useful for genetic screens and for the insertion of genetic and molecular markers at particular points in the genome to identify interesting genes based on their mutant phenotypes.

Factors affecting the activity of restriction enzymes

Depending on the substrate DNA and the reaction conditions, restriction enzymes show a wide variation of cleavage and possible star activity. In order to obtain the desired cleavage, it becomes important to control the following factors:

  1. Star activity: Under sub-optimal reaction conditions, some restriction enzymes cleave base sequences at sites different from the defined recognition sequence. In other words, they cleave at non-specific sites. This phenomenon is called star activity. Some of the factors that induce star activity are high salt and glycerol concentration, presence of impurities, excessive enzyme compared to substrate DNA, increased incubation time, or incompatible buffer and cofactor.
  2. Methylated DNA: Several DNA molecules are methylated at the recognition site, making them resistant to cleavage by certain restriction enzymes. For example, most E. coli strains express Dam or Dcm methyltransferases that methylate specific recognition sites to form G6mATC and C5mCA/TGG, respectively. G6mATC is resistant to cleavage by Mbo I.
  3. Temperature: Most endonucleases optimally digest the target DNA at 37 °C. However, there are some exceptions with lower or higher optimal temperatures. For example, Taq I (Catalog No. R9507) optimally digests at 65 °C and Apa I (Catalog No. R4258) digests at 25 °C.

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