The two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated in the year 1963. One was the enzyme which added methyl groups to DNA, while the other cut DNA. The latter was called restriction endonuclease.
A restriction enzyme or restriction endonuclease is an enzyme that cleaves DNA into fragments at or near specific recognition sites within the molecule known as restriction sites. Based on their mode of action restriction enzymes are classified into Exonucleases and Endonucleases.
a. Exonucleases are enzymes which remove nucleotides one at a time from the end of a DNA molecule. e.g. Bal 31, Exonuclease III.
b. Endonucleases are enzymes which break the internal phosphodiester bonds within a DNA molecule. e.g. Hind II, EcoRI, Pvul, BamH
Molecular scissorsThe restriction enzymes are called as molecular scissors. These act as foundation of recombinant DNA technology. These enzymes exist in many bacteria where they function as a part of their defence mechanism called restriction-modification system.
There are three main classes of restriction endonucleases: Type I, Type II and Type III, which differ slightly by their mode of action. Only type II enzyme is preferred for use in recombinant DNA technology as they recognise and cut DNA within a specific sequence typically consisting of 4-8 bp.
The restriction enzyme Hind II always cut DNA molecules at a point of recognising a specific sequence of six base pairs. This sequence is known as recognition sequence. Today more than 900 restriction enzymes have been isolated from over 230 strains of bacteria with different recognition sequences. This sequence is referred to as a restriction site and is generally –palindromic which means that the sequence in both DNA strands at this site read same in 5’ – 3’ direction and in the 3’-5’ direction.
Restriction endonucleases are named by a standard procedure. The first letter of the enzymes indicates the genus name, followed by the first two letters of the species, then comes the strain of the organism and finally a roman numeral indicating the order of discovery. For example, EcoRI is from Escherichia (E) coli (co), strain RY 13 (R) and first endonuclease (I) to be discovered.
The exact kind of cleavage produced by a restriction enzyme is important in the design of a gene cloning experiment. Some cleave both strands of DNA through the centre resulting in blunt or flush end. These are known as symmetric cuts. Some enzymes cut in a way producing protruding and recessed ends known as sticky or cohesive end. Such cut are called staggered or asymmetric cuts.