What is Restriction Endonuclease?

Restriction endonuclease, also known as restriction enzyme, is a class of enzymes isolated from bacteria by W. Arber, H. Smith and D. Nathans et al. (1979). Restriction enzymes have extremely high specificity. They recognize specific sites on double-stranded DNA and cut both strands to form sticky or blunt ends. Restriction enzymes can be used to dissect delicate DNA molecules, and are indispensable tools in the analysis of chromosome structure, DNA restriction enzyme maps, determination of longer DNA sequences, gene isolation, and in vitro recombination of genes.

The biological function of restriction enzymes is to defend or limit the extraneous DNA (such as phage DNA) that invades the cell. Prokaryotes use their unique restriction enzymes to cut foreign DNA into non-infectious fragments. But it cannot degrade the chromosomal DNA in its own cells, because there are co-located DNA methylases in the cell to modify the corresponding sequence to protect it.

Restriction enzymes can be divided into three types. Type I and type III restriction enzymes need to consume ATP to hydrolyze DNA. Some subunits of the holoenzyme have the activity of chemically modifying DNA by adding methyl groups to special bases. Type I restriction endonuclease cuts DNA at a random site; Type III restriction enzyme recognizes the specific nucleotide sequence of double-stranded DNA and cuts the double-stranded DNA at or near this site.

Type II restriction endonucleases have been widely used in the cloning and sequence analysis of DNA molecules, because they do not require ATP to hydrolyze DNA, and they do not modify DNA by methylation or other ways. The most important thing is that they cut the DNA strand within or near the specific nucleotide sequence they recognize. These special sequences often contain 4 or 6 nucleotide residues, usually have a palindromic structure, and form sticky ends (or blunt ends) after cutting. For example, a restriction enzyme in Escherichia coli is called EcoR I, which recognizes the following hexanucleotide sequence:
5'……GAATTC……3'

3'……CTTAAC……5'
The two chains of this palindrome have the same structure when reading the sequence in the 5'→3' direction. When EcoR I encounters this sequence of DNA, it will stagger the cut of this DNA strand, resulting in a protruding 5'end.

5'……G AATTC……3'

3'……CTTAA C……5'

Some restriction enzymes, such as Pst I, recognize the sequence 5'-CTGCAG-3' and cut the DNA double strand between A and G to produce 3'overhanging sticky ends. Some restriction enzymes, such as Bal I, produce DNA fragments with blunt ends in the DNA strand.

The naming of restriction enzymes is special. Take EcoR I as an example. The first capital letter E is the first letter of the genus name of Escherichia coli E. coil, and the second and third lowercase letters co are the first two letters of its species name. Use a capital R for the fourth letter to indicate the strain of E. coli used. The last Roman character indicates the number of this type of enzyme isolated from the bacterium. At present, there are many purified restriction enzymes. There are about 100 kinds of commonly used restriction enzymes, and they have been transformed into commercial products, which greatly saves researchers' time.