Introduction:
A restriction enzyme is a nuclease enzyme that breaks down DNA sequences at random or specified recognition regions known as restriction sites. In bacteria, restriction enzymes interact with modification enzymes to form a combination system (restriction + modification system) that methylates the bacterial DNA. Methylation of bacterial DNA at the recognition sequence often protects the bacteria’s own DNA from cleavage by restriction enzymes.
There are two different kinds of restriction enzymes:
- Exonucleases catalyzes hydrolysis of terminal nucleotides from the end of DNA or RNA molecule either 5’to 3’ direction or 3’ to 5’ direction. Example: exonuclease I, exonuclease II, etc.
- Endonucleases can recognize specific base sequence (restriction site) within DNA or RNA molecule and cleave internal phosphodiester bonds within a DNA molecule. Example: EcoRI, Hind III, BamHI etc.
Endonucleases are the enzymes that produce internal cuts, called cleavage that break the phosphodiester bonds in the polynucleotide chain. Some endonucleases cleave only one of the strands of a DNA duplex, e.g., S1 nuclease (from Aspergillus oryzae); such cuts are usually known as nicks.
W. Arber proposed the presence of restriction enzymes in the 1960s, and the first actual restriction endonuclease was discovered in 1970. Smith, Nathans, and Arber were awarded the Nobel Prize in Physiology and Medicine in 1978 for discovering endonucleases.
In contrast many endonucleases cleave both the strands of DNA molecules; many of such endonucleases DNA molecules at a random site, e.g., deoxyribonucleases I (DNase I that is obtained from a cow pancreas). But a class of endonucleases cleaves known as restriction endonucleases, and the sites recognized by them are called recognition sequences or recognition sites. The recognition sequences differ and are distinct to each restriction endonuclease or restriction enzyme. By the year 2000, over 1,200 distinct restriction enzymes had been identified.
The phenomenon of bacteriophage host restriction resulted in the discovery and naming of restriction enzymes. The restriction on phage host range is due to the presence of restriction enzymes in the host cells, which recognizes and cleaves foreign DNA introduced into the cell. The DNA of a cell is protected from its own endonucleases by methylation (usually A and C) within their recognition sites. Thus, DNA molecules with the same methylation pattern as a bacterial cell will be recognized as their own DNA, while those without it will be considered foreign DNA.
Types of Restriction of Endonuclease:
There are the following distinct types of restriction endonuclease
Type I restriction endonuclease
These are complex endonuclease and have recognition sequences of about 15 bp; they cleave the DNA about 100bp away from the 5’-end of the sequence “TCA” located within recognition site e.g., ecoK, EcoB, etc.
Type II restriction endonuclease
These are remarkably stable and induce cleavage either, in most cases, within or immediately outside their recognition sequences, which are symmetrical. More than 350 different type II endonuclease with over 100 different recognition sequences are known. They require mg 2+ ions form cleavage. The first type II enzyme to be isolated was hindII in 1970. Only type II restriction endonucleases are used for restriction mapping and gene cloning in view cleavage only at specific sites.
Type III restriction endonuclease
These are intermediate between the type I and type II enzymes; they cleave DNA in the immediate vicinity of their recognition sites, e.g., EcoP1, EcoP15, HindfII, etc. Type I and Type III restriction enzymes are not used in gene cloning because random cutting is not useful for gene cloning. The type III enzymes recognize asymmetric target sites, and cleave the DNA duplex on one side of the recognition sequences up to 20bp away.
Applications:
Restriction endonucleases are essential for DNA cloning and sequencing. They act as tools for cutting DNA molecules at preset locations, which is a prerequisite for gene cloning or recombinant DNA technology.