2-1.4.3 Type III restriction enzymes:

• These enzymes recognize and methylate the same DNA sequence but cleave 24–26 bp away.

• They have two different subunits, in which one subunit (M) is responsible for recognition and modification of DNA sequence and other subunit (R) has nuclease action.

• Mg⁺² ions, ATP are needed for DNA cleavage and process of cleavage is stimulated by SAM.

• Cleave only one strand. Two recognition sites in opposite orientation are necessary to break the DNA duplex.

Property	Type I RE	Type II RE	Type III RE
Abundance	Less common than Type II	Most common	Rare
Recognition site	Cut both strands at a non- specific location > 1000 bp away from recognition site	Cut both strands at a specific, usually palindromic recognition site (4-8 bp)	Cleavage of one strand, only 24-26 bp downstream of the 3´ recognition site
Restriction and modification	Single multifunctional enzyme	Separate nuclease and methylase	Separate enzymes sharing a common subunit
Nuclease subunit structure	Heterotrimer	Homodimer	Heterodimer
Cofactors	ATP, Mg²⁺ , SAM	Mg²⁺	Mg²⁺ (SAM)
DNA cleavage requirements	Two recognition sites in any orientation	Single recognition site	Two recognition sites in a head-to-head orientation
Enzymatic turnover	No	Yes	Yes
DNA translocation	Yes	No	No
Site of methylation	At recognition site	At recognition site	At recognition site

Table 2-1.4: Comparative properties of restriction enzymes