Restriction enzymes or restriction endonucleases bind to a specific double-stranded DNA sequence and cleave the DNA at a specific site. These enzymes are produced in bacteria as a part of their defense system against viruses. Type II restriction enzymes are most useful for recombinant DNA technology as they cleave within or at short specific distances from a recognition site. They do not use ATP for their activity and require Mg2+ as a cofactor.
Analysis of the ECORV Endonuclease active site showed that the Mg2+ is bound to six ligands. They are 3 water molecules (H2O), 2 carboxylates of the enzyme’s aspartate amino acid, and 1 oxygen atom of the phosphoryl group present on the cleavage site. The Mg2+ ion activates a water molecule and positions it in the active site of the enzyme so that it can attack the phosphate on the nucleotide.