Restriction enzymes (also known as restriction endonucleases) are a group of bacterial enzymes which cut double-stranded DNA (dsDNA) into smaller fragments at specific points. They are a defence mechanism used by bacteria to cleave the DNA of invading viruses, thereby restricting their expression. The exploitation of restriction enzymes ability to cut large pieces of DNA into smaller fragments (called restriction fragments) and the highly specific way in which they do this has played a crucial role in the exponential advancement of biotechnology in recent decades.
Restriction enzymes cut DNA at specific places by recognising short DNA sequences called restriction sites. These sequences are typically 4-8bp long and are specific to each enzyme. Restriction sites can be found on any DNA molecule, whether it be viral DNA, fish DNA, human DNA etc. This is an important aspect in biotechnology as it means genomic DNA from any species of interest can be cut into smaller, more manageable junks. However, this does pose the question: how do bacteria prevent their own DNA form being cleaved by their restriction enzymes?
Bacteria methylate their own restriction sites to overcome this problem. The addition of a methyl group prevents restriction enzymes form binding and cleaving the DNA. It allows the bacteria to identify self from non-self. This is an efficient tactic employed by bacteria as restriction sites are not only found in all types of DNA but they all share a similar design wherever they are found.
Most restriction sites are palindromic. They exhibit twofold rotational symmetry. This means that the two strands within the restriction site will have the same sequence if read in a 5’-3’ direction (Diagram 1). Different restriction enzymes will act upon different palindromic sequences but the nature by which they cut the DNA is always the same.
Restriction enzymes cut the DNA backbone by hydrolysis of the phosphodiester bond. This results in