Restriction enzymes, or restriction endonucleases, are a broad class of DNA-cutting enzymes that occur naturally in bacteria and are instrumental in its defense against viral infections (i.e. bacteriophages). Considered as nature’s molecular scissors, restriction enzymes recognize specific nucleotide sequences of DNA, called restriction sites, on which to bind and subsequently cleave into fragments via hydrolysis of the phosphodiester backbone. Restriction fragments can possess either blunt ends, which have 5'-phosphate groups that promote ligation, or sticky ends, which have 3'- or 5'-overhangs of 1 to 4 nucleotides and are more cohesive. These small stretches of single-stranded DNA can self-ligate or ligate with a complementary region on another DNA molecule. Since fragmentation occurs at or near the recognition site and transpires in a predictable manner, restriction enzymes have become essential tools in genetic engineering (e.g. DNA cloning and DNA fingerprinting).

Types of Restriction Enzymes

Different bacterial species produce restriction enzymes that recognize and cut different nucleotide sequences. To this date, scientists have identified and purified hundreds of restriction enzymes, and have categorized them into four groups according to their composition, the characteristics of the cleavage site, and the enzyme cofactor requirements.

• Type I restriction enzymes cleave randomly at sites around 1000 bp away from the restriction site. ATP, AdoMet and Mg²⁺ are required as enzyme cofactors. Common examples of Type I restriction enzymes include EcoK I, EcoA I and CfrA I.
• Type II restriction enzymes cleave within or a short specific distances away from the restriction site, and require Mg²⁺ as an enzyme cofactor. Common examples of Type II restriction enzymes include EcoR I, BamH I and Hind III.
• Type III restriction enzymes cleaves at sites 24 to 26 bp away from the restriction site, and require ATP and Mg²⁺ as enzyme cofactors. Common examples of Type II restriction enzymes include EcoP I, EcoP15 I and Hinf III.
• Type IV restriction enzymes cleave within or at short specific distances from the restriction site. They also target modified DNA such as methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA.