Everything You Want to Learn About Restriction Enzymes

If there were no restriction enzymes, what would be the development of molecular biology research today? For 40 years, restriction endonucleases, working behind the scenes, have quietly promoted many basic biological research and commercial applications. Restriction endonucleases (also known as restriction enzymes) were first found in bacteria, but later they were also found in some archaea. Usually, restriction endonucleases cut double-stranded DNA. Each restriction endonuclease will recognize a specific DNA sequence. Depending on the type of endonuclease, the DNA can be cut within the recognition sequence or not far from the recognition sequence. The length of the recognition sequence is usually 4-8 bp, and after digestion, sticky ends (5' or 3'overhangs) and blunt ends will be formed. Today, about 4000 restriction enzymes have been identified in the industry, of which more than 600 are commercially available.

The history of restriction endonucleases

In the early 1950s, many research teams observed differences in the infection efficiency of bacteriophages against different bacterial host strains of the same species. It was not until the 1960s that people discovered the mechanism by which the host controls mutation, which was related to the digestion of phage DNA, and then discovered and isolated restriction enzymes. With pioneering research on restriction enzymes, Daniel Nathans, Hamilton Smith and Werner Arber jointly won the 1978 Nobel Prize in Physiology or Medicine. With the discovery of DNA ligase (a kind of DNA modifying enzymes) and the growing family of site-specific restriction endonucleases, recombinant DNA technology emerged.

Restriction enzyme naming rules

This naming rule takes into account the three characteristics of the source of endonuclease: genus name, species name and strain or serotype. Together they form a short name, followed by Roman numerals, representing multiple restriction enzymes from the same strain. For example, HindIII (or Hind III according to earlier naming conventions) stands for:

"H" stands for Haemophilus

"In" stands for influenzae

"D" stands for serotype d

"III" is used to distinguish other restriction enzymes from Haemophilusinfluenza serotype d (for example, HindII and HindIII).

Classification of restriction enzymes

According to the complexity of the structure, recognition sequence, cleavage site location and cofactor requirements, restriction endonucleases are divided into four categories: Type I, Type II, Type III, and Type IV.

1. Features of Type I:

a) A multi-subunit protein with both restriction and methylation activity.

b) It needs ATP.

c) The distance between the cutting site and the recognition site is variable.

2. Features of Type II:

a) Specific recognition sequence

b) The cleavage site is located within or adjacent to the recognition sequence

c) It generates 5? phosphate and 3? hydroxyl ends at the cleavage site

d) It requires Mg2+

3. Features of Type III

a) It consists of two recognition sequences in opposite directions

b) The distance between the cleavage site and one of the recognition sequences is constant

c) It needs ATP

4. Features of Type IV

a) It only cuts methylated DNA

b) The cutting site is approximately 30 bp away from the recognition site

Specificity of recognition site and cleavage

Another important method for classifying and comparing restriction enzymes is isoschizomer and heteroschizomer.

Isoschizomers are restriction endonucleases with the same recognition sequence and the same specificity. For example, AgeI and BshT1 can be identified and cut 5'-A?CCGGT-3' through the same map. However, a series of isoschizomers may have differences in preferred sites, reaction conditions, methylation sensitivity and star activity.

Heterochlease can recognize the same nucleotide sequence, but will cut DNA at different sites. For example, both SmaI (5'-CCC?GGG-3') and XmaI (5'-C?CCGGG-3') can recognize 5'-CCCGGG-3'. However, the cutting method is different, resulting in different types of ends (in this case, SmaI produces blunt ends, and XmaI produces 5? protruding ends).

The difference in recognition sequence and cleavage map makes restriction endonucleases an extremely flexible and powerful tool for identification and manipulation of genetic material.

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