2-1.4 Classification of Restriction Endonucleases:

There are three major classes of restriction endonucleases based on the types of sequences recognized, the nature of the cut made in the DNA, and the enzyme structure:

•  Type I restriction enzymes

•  Type II restriction enzymes

•  Type III restriction enzymes

2-1.4.1 Type I restriction enzymes:

•  These enzymes have both restriction and modification activities. Restriction depends upon the methylation status of the target DNA.

•  Cleavage occurs approximately 1000 bp away from the recognition site.

•  The recognition site is asymmetrical and is composed of two specific portions in which one portion contain 3–4 nucleotides while another portion contain 4–5 nucleotides and both the parts are separated by a non-specific spacer of about 6–8 nucleotides.

•  They require S-adenosylmethionine (SAM), ATP, and magnesium ions (Mg²⁺) for activity.

•  These enzymes are composed of mainly three subunits, a specificity subunit that determines the DNA recognition site, a restriction subunit, and a modification subunit

 

2-1.4.2 Type II restriction enzymes:

•  Restriction and modification are mediated by separate enzymes so it is possible to cleave DNA in the absence of modification. Although the two enzymes recognize the same target sequence, they can be purified separately from each other.

•  Cleavage of nucleotide sequence occurs at the restriction site.

•  These enzymes are used to recognize rotationally symmetrical sequence which is often referred as palindromic sequence.

•  These palindromic binding site may either be interrupted (e.g. BstEII recognizes the sequence 5´-GGTNACC-3´, where N can be any nucleotide) or continuous (e.g. KpnI recognizes the sequence 5´-GGTACC-3´).

•  They require only Mg²⁺ as a cofactor and ATP is not needed for their activity.

•  Type II endonucleases are widely used for mapping and reconstructing DNA in vitro because they recognize specific sites and cleave just at these sites .