Restriction Endonucleases
There are four different types of restriction endonucleases or restriction-modification (R-M) system viz. type I, type II, type III and type IIs (Table). The type I restriction endonucleases were the first to be characterized. The recognition sequences are long and are not palindromic. The enzyme cleaves at unmodified DNA away from the recognition sequence. Sometimes, the target DNA may be methylated before it is cut as the same enzyme performs methylation. Due to these features, type I restriction endonucleases were not considered appropriate for gene manipulation experiments.
Important: Palindromic sequences are the sequences that read the same when read from either 5' to 3' or from 3' to 5'. For example, the recognition sequence of EcoRI is:
The above sequence when read from 5' to 3' or from 3' to 5' is the same. Hence, the recognition sequence for EcoRI is palindromic.
Table: Types of restriction endonucleases
Type of restriction endonucleases |
Characteristics |
Examples |
|
Type I |
It is one enzyme with different subunits one each for recognition, cleavage, and methylation. It recognizes and methylates a single sequence but makes the cut 1000 bp away. |
EcoK, EcoB |
|
Type II |
These are two different enzymes but recognize the same recognition sequence, which is usually palindromic. The two enzymes either cleave or modify the recognition sequence. |
EcoRI, HindIII, HaeII etc |
|
Type III |
It is one enzyme with two different subunits: one subunit for recognition and modification and other for cleavage. It recognizes and methylates the same sequence but makes the cut 24-26 bp away. |
|
|
Type IIs |
These are two different enzymes but recognition sequence is not palindromic. The cut is made on one side of the recognition sequence up to 20 bp away. |
|
Of all the types of restriction endonucleases known, type II are the most useful and they have many advantages over type I and type III. First advantage, the restriction and modification functions are performed by separate enzymes so it is possible to cleave the DNA in the absence of modification. Second advantage, the restriction activity of the enzymes does not require cofactors like ATP or S-adenosylmethionine, so they can be easily used. Third advantage, type II enzymes recognize a specific sequence, usually palindromic, within a DNA molecule and cut within the sequence. Most of the type II enzymes make sticky o staggered cuts which are more useful in recombinant DNA technology. Further, type IIs enzymes are also similar to type II enzymes but they make the cut at a distance from the recognition sequence which limits their application.