Restriction enzyme

A restriction enzyme (also restriction endonuclease) is an enzyme, usually synthesised by bacteria, that selectively cuts up DNA (single or double-stranded) in a process called 'restriction'. The restriction system originally evolved as a defence mechanism against foreign DNA; the bacterium's own genomic DNA is protected from digestion by the methylation of bases.

Restriction enzymes recognise DNA at a specific sequence called the recognition site and then cut the DNA at a restriction site; these may or may not be at the same location in the molecule. The restriction site of enzymes varies to produce either blunt or sticky ends. Sticky ends result in a 3' or 5' overhang at the site of cleavage, while blunt ends do not result in any such overhang. Sticky ends are often useful in recombinant technology as they provide implicit complementarity between two molecules.

When generating a recombinant molecule, it is common to cut the desired complementary sequences with two different restriction enzymes, for instance:

[EcoR1] target DNA [BamH1]
[EcoR1] plasmid DNA [BamH1]

Because these enzymes produce different sticky ends, it ensures that the target DNA is inserted into the plasmid with the desired orientation. This is called directional cloning.

Restriction enzymes come in three types:

Type I: these are complex and have both restriction and modification functions. Type I restriction enzymes cleave DNA randomly and at some distance from their recognition site; as such they are not useful to genetic engineers.

Type II: these cleave DNA at defined and predictable locations within or near the recogniton site. The recognition site is usually palindromic (i.e. reads identically in 5' to 3' orientation on both strands). These are the most commonly used in molecular biology.

Type III: these are also complex, restriction-and-modification enzymes. They invariably cleave outside of their recognition site. These are occasionally useful in recombinant technology; DNA is not cleaved within the molecule.

Restriction enzymes are named by the species and strain from which they were isolated. For instance, EcoR1, a common restriction enzyme, is named after E. coli, strain R, isolate 1 since it was the first restriction enzyme to be isolated from strain R of E. coli.

More important factors regarding restriction enzymes:

  • The size of a restriction site varies, including 4, 5, 6, 10 and 12bp sites
  • Some restriction enzymes have degenerate recognition sites, such as HincII, which recognises GTY/RAC where Y is a variable pyrimidine and R a variable purine
  • Some enzymes have different restriction sites but generate the same sticky ends (these are isocaudomers)
  • There are 2500 restriction enzymes and only 350 recognition sites: enzymes which have the same recognition site are isoschizomers
  • An enzyme that recognizes the same sequence but cuts it differently is a neoschizomer
  • The frequency with which a restriction enzyme will cleave DNA depends upon the length of its restriction site: for instance a 4bp recognition site gives a cleavage frequency of 44 = every 256 base pairs