Restriction Endonucleases

One of the first type II restriction endonucleases to be isolated was from the bacterium Escherichia coli and was named EcoRI. The use of italics for naming restriction enzymes has been abandoned lately, hence this book will follow the same. EcoRI is a homodimeric proteini.e. made up of two identical proteins. EcoRI binds to a specific sequence (5'- GAATTC- 3') within the DNA and its recognition sequence consists of 6-bp and the cleavage occurs between G and C residue in each strand. The cleavage refers to the breaking of phosphodiester bond between G and C within the recognition sequence which produces staggered or sticky ends. In addition to EcoRI, more than 3700 restriction enzymes, with over 250 recognition sequences, have been isolated from various host bacteria and more than 300 are in use in the laboratory.

Table 1: some of the common restriction endonucleases 

If a restriction enzyme has a 7-bp restriction sequence, it will cut the DNA approximately on an average at, 4⁷= 16,384 bp. Similarly, if the restriction site is 6-bp or 8-bp long, the enzyme will cut the DNA once in every 4⁶=4096 bp and 4⁸=65,536 bp respectively. The restriction mapping of large DNA fragments can be done by using cutters with 7-bp or 8-bp recognition sequences. However, such enzymes are less in number.

Type IIs restriction endonucleases are a subgroup of type II restriction endonucleases. They are sometimes used in cloning and other molecular biology studies. these enzymes cut the DNA a fixed number of nucleotides away from one end of the recognition sequence. These enzymes produce sticky ends after cleavage. An example of type IIs enzyme is FokI whose recognition sequence is and cuts the nucleotide sequence 9 bp downstream the upper strand and 13 nucleotides downstream the lower strand. This information can be represented as: where N represents A, T, C or G. this representation also denotes that the nucleotides (N) opposite to each other are base-paired. This information can also be represented as or . Some examples of type IIs restriction enzymes have been given in Table.

Table 2: Some common type II restriction endonucleases 

Sometimes, interesting correlations can be observed between the recognition sequences of different restriction endonucleases. Some enzymes give different cuts within same recognition site. For example, the restriction enzymes XmaI and SmaI, both obtained from different organisms have same recognition sequence but give different cuts as follows:

The first enzyme discovered is known as the prototype while the subsequent enzymes are known as isoschizomers. Sometimes, a recognition sequence is identified by several restriction endonucleases and each of them cuts at a different site within the recognition sequence to give different cleavage products. Such enzymes are called as neoschizomers. For example, the recognition sequence is identified by restriction endonucleases like KasI, NarI, SfoI, MchI, BinSII, BbeI, NdaI, MIy113I, DinI, EgeI and EheI. Further, there are enzymes which have different recognition sequences but produce same nucleotide extensions. Such enzymes are called as isocaudomers. For example, BamHI and Sau3AI.

There are enzymes available which can be used to ascertain if the  recognition sequence is methylated or not. Some restriction enzymes will cleave the DNA only if the cytosine residues within the recognition sequences are methylated while some will cleave the DNA only if they are not methylated. For example, HpaII will cleave the recognition sequence

only if cytosine residues are not methylated while MspI, an isoschizomer of HpaII, will cleave this sequence in both the conditions i.e. with methylated cytosine residues and non-methylated residues. If any DNA molecule is cut by MspI but not cut by HpaII, then the recognition site is methylated while if both the enzymes cut the DNA molecule then the recognition site is not methylated.