One of the most important steps in molecular biology, especially molecular genetics and analysis, is the isolation of DNA from the human genome and make many copies of it. Now, these copies can be utilized for further analysis of whatsoever type.
A key event in the development of molecular genetics methodology has been the discovery of Restriction Enzymes, also known as Restriction Endonucleases.
What is Restriction enzyme?
A restriction enzyme is a protein isolated from bacteria that cleaves DNA sequences at sequence-specific sites, producing DNA fragments with a known sequence at each end. The use of restriction enzymes is critical to certain laboratory methods, including recombinant DNA technology and genetic engineering.
The restriction enzymes protect the live bacteria from bacteriophages. They recognize and cleave at the restriction sites of the bacteriophage and destroy its DNA.
Restriction enzymes are important tools for genetic engineering. They can be isolated from the bacteria and used in the laboratories.
The restriction enzymes recognize short and specific nucleotide sequences in the DNA known as the recognition sequences. When the restriction enzyme recognizes a DNA sequence, it hydrolyzes the bond between adjacent nucleotide and cuts through the DNA molecule.
The bacteria prevents its own DNA sequences from degradation by the addition of the methyl group at the adenine or cytosine bases within the recognition sequence with the help of enzyme methylases.
Types of Restriction Enzymes
There are two different kinds of restriction enzymes:
Exonucleases: restriction exonucleases are primarily responsible for hydrolysis of the terminal nucleotides from the end of DNA or RNA molecule either from 5’ to 3’ direction or 3’ to 5’ direction; for example- exonuclease I, exonuclease II, etc.
Endonuclease: restriction endonucleases recognize particular base sequences (restriction sites) within DNA or RNA molecule and catalyze the cleavage of internal phosphodiester bond; for exEcoRI, Hind III, BamHI, etc.
Based on the composition, characteristics of the cleavage site, and the cofactor requirements, the restriction endonucleases are classified into four groups, Type I, II, III, and IV.
#1. Type I Restriction Enzyme
These restriction enzymes cut the DNA far from the recognition sequences. However, they do not produce discrete restriction fragments, hence, are of not much practical value.
These are complex, multi-subunit restriction and modification enzymes. They were initially thought to be rare, but through genomic analysis, they are found to be common and are of considerable biochemical interest.
#2. Type II Restriction Enzyme
These enzymes cut at specific positions closer to or within the restriction sites. Discrete restriction fragments and gel banding patterns are observed. They are exclusively used for DNA analysis and gene cloning in the laboratories.
These are a family of unrelated proteins. They are named after the bacterial species from which they are isolated. For eg., EcoRI is isolated from bacterial species E.coli.
The restriction enzymes generate two different types of cuts. Blunt ends are produced when they cut the DNA at the centre of the recognition sequence, and sticky ends produce an overhang.
#3. Type III Restriction Enzyme
These are multi-functional proteins with two subunits- Res and Mod. It is a modification methyltransferase. The DNA sequence specific for the system is recognized by the Mod subunit.
The type III enzymes recognize and methylate the same DNA sequence. However, they cleave nearly 24-26 base pairs away.
They are composed of two different subunits. The recognition and modification of DNA are carried out by the first subunit- ‘M’ and the nuclease activity is rendered by the other subunit ‘R’.
DNA cleavage is aided by ATP as well as Mg2+ whereas SAM is responsible for stimulating cleavage.
Only one of the DNA strand is cleaved. However, to break the double-stranded DNA, two recognition sites in opposite directions are required.
#4. Type IV Restriction Enzyme
Type IV enzymes recognize modified, typically methylated DNA and are exemplified by the McrBC and Mrr systems of E. coli.
Star Activity
Some restriction enzymes are capable of cleaving recognition sites which are similar to but not identical to the defined recognition sequence under non-standard reaction conditions (low ionic strength, high pH).
Isoschizomers, Neoschizomers, and Isocaudomers
- Isoschizomers are the restriction enzymes which recognize and cleave at the same recognition site. For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other.
- Neoschizomers are the restriction enzymes which recognize the same site and have a different cleavage pattern. For example, SmaI (GGG/CCC) and XmaI (G/GGCCC) are neoschizomers of each other.
- Isocaudomers are the restriction enzymes which recognize slightly different sequences but produce the same ends. For example, both Sau3a and BamHI render a 5’-GATC-3’ sticky end although both have different recognition sequences.
Applications Of Restriction Enzyme
- Restriction enzymes are utilized for gene insertion into plasmids during cloning and protein expression experiments.
- They are also used for SNPs analysis and identifying gene alleles. However, this is only possible if a mutation alters the restriction site of the enzyme.
- REs are used for the Restriction Fragment Length Polymorphism (RFLP) analysis for identifying strains or individuals of particular species.