Restriction Enzymes – Molecular Scalpels

The biological function of restriction enzymes is derived from the defense of foreign DNA from invading cells. The restriction-modification system of bacteria makes the DNA bases of bacteria to be methylated and modified, so that they are not recognized and cut by their own restriction enzymes.

The development of restriction endonucleases
In the early 1960s, Werner Arber came up with the idea that there was an enzyme that would cut the DNA of a virus, thereby limiting the virus' proliferation.

Kent Wilcox and Hamilton Smith discovered the first type II restriction endonuclease Hind II, making the restriction enzymes fully utilized. Hind II can process DNA precisely, and they also found that methylase can protect the original DNA of bacteria from Hind II's attack by methylation modification.

Kathleen Danna and Daniel Nathans obtained the restriction endonuclease map by studying the specific cleavage of SV40 DNA by Haemophilus influenzae restriction enzymes.

Daniel Nathans, Hamilton Smith and Werner Arber shared the 1978 Nobel Prize in Physiology or Medicine for their pioneering research in the field of restriction enzymes.

Up to now, scientists have isolated a variety of restriction enzymes from prokaryotes. Restriction enzymes have become important "scalpels" in genetic engineering, and have been commercialized and widely used in genetic engineering.

Classification of restriction endonucleases
Restriction enzymes can be divided into three types, namely type I, type II, and type III, according to factors such as subunit composition, recognition site, restriction enzyme cleavage position, cofactor, and mode of action.

Type I and type III restriction enzymes do not have practical value because they cannot produce specific cleavage fragments. Type II restriction enzymes have been widely used in DNA molecular cloning and sequence analysis because they do not require ATP to hydrolyze DNA and do not methylate or otherwise modify DNA. And they cut double-stranded DNA within or near the recognition sequence.

The cleavage sequence of type II restriction endonucleases is often a 4-6 bp palindrome. The enzyme cuts in two ways: one is staggered, creating sticky ends, and the other is cleaving double strands at the same location, creating blunt ends.

Type II restriction enzymes can be further subdivided into sub-categories such as IIP, IIA, IIB, IIC, and IIS. Among them, type IIS restriction enzymes can cleave DNA duplexes at a specific distance downstream from one side of its asymmetric recognition site, since the recognition sequence and catalytic region of such restriction enzymes are separated by a linking polypeptide.

A prominent feature of type IIS restriction enzymes is that there is no requirement for the sequence of the cleavage site, that is, any nucleotide sequence outside the sequence can be recognized. According to this characteristic, during the in vitro synthesis of mRNA, the Poly(A) tail can be obtained by restriction cleavage and transcription from the plasmid DNA template, which can add a specific number of Poly(A) to the mRNA product.

Factors Affecting Restriction Endonuclease Activity
(1) DNA purity: Impurities in DNA such as protein, phenol, chloroform, ethanol, SDS, EDTA, etc. will affect the activity of the enzyme, which is generally improved by purifying DNA, increasing the amount of enzyme, prolonging the incubation time and expanding the reaction system.
(2) Degree of DNA methylation: Escherichia coli generally has two methylase-modified plasmids, dam and dcm, and strains with methylase-inactivating mutations must be used in genetic engineering.
(3) Temperature: The optimal reaction temperature of different restriction enzymes is different. Most are 37˚C, with a few restriction enzymes having an optimum temperature of 40-65˚C.
(4) Buffer: Buffer is an important factor affecting the activity of restriction enzymes. Commercial restriction enzymes generally come with special buffers.