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.
A restriction enzyme is a kind of nuclease enzyme which is capable of cleaving double-stranded DNA. The enzymes may cleave DNA at random or specific sequences which are referred to as restriction sites. The recognition sites are palindromic in origin, that is, they are the sequences which are read the same forward and backward.
These restriction enzymes are produced naturally by bacteria. The bacterial species use it as a form of defense mechanism against viruses. However, in bacteria, restriction enzymes are present as a part of a combined system called the restriction modification system. The bacterial species modify their own DNA with the help of enzymes which methylate it. This particular process of methylation of bacterial DNA protects it from cleavage from its own restriction endonucleases.
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.
The first restriction enzyme to be discovered was Hind II in the year 1970. In 1978, Daniel Nathans, Werner Arber, and Hamilton O. Smith were awarded the Nobel Prize for Physiology or Medicine.
Restriction Enzyme Nomenclature
The very name of the restriction enzymes consists of three parts:
- An abbreviation of the genus and the species of the organism to 3 letters, for example- Eco for Escherichia coli identified by the first letter, E, of the genus and the first two letters, co, of the species.
- It is followed by a letter, number or combination of both of them to signify the strain of the species.
- A Roman numeral to indicate the order in which the different restriction-modification systems were found in the same organism or strain per se.
Classification of Restriction Endonucleases
Based on the types of sequences identified, the nature of cuts made in the DNA, and the enzyme structure, there are three classes:
- Type I restriction enzymes,
- Type II restriction enzymes, and
- Type III restriction enzymes.
A. Type I Restriction Enzymes
- Type I restriction enzymes possess both restriction and modification activities. In this case, the restriction will depend upon the methylation status of the target DNA sequence.
- Cleavage takes place nearly 1000 base pairs away from the restriction site.
- The structure of the recognition site is asymmetrical. It is composed of 2 parts. One part of the recognition site is composed of 3-4 nucleotides while the other one contains 4-5 nucleotides. The two parts are separated by a non-specific spacer of about 6-8 nucleotides.
- For their function, the type I restriction enzymes require S- adenosylmethionine (SAM), ATP, and Mg2+
- They are composed of 3 subunits, a specificity subunit which determines the recognition site, a restriction subunit, and a modification subunit.
B. Type II Restriction Enzymes
- Two separate enzymes mediate restriction and modification. Henceforth, DNA can be cleaved in the absence of modifying enzymes. Although the target sequence identified by the two enzymes is the same, they can be separately purified from each other.
- The nucleotides are cleaved at the restriction site only. The recognition sequence is rotationally symmetrical, called palindromic sequence. The specific palindromic site can either be continuous (e.g., KpnI identifies the sequence 5´-GGTACC-3´) or non-continuous (e.g., BstEII recognizes the sequence 5´-GGTNACC-3´, where N can be any nucleotide)
- These require Mg2+ as a cofactor but not ATP.
- They are required in genetic mapping and reconstruction of the DNA in vitro only because they identify particular sites and cleave at those sites only.
How they work:
- The type II restriction enzymes first establish non-specific contact with DNA and bind to them in the form of dimmers.
- The target sequence is then detected by a combination of two processes. Either the enzyme diffuses linearly/ slides along the DNA sequence over short distances or hops/ jumps over long distances.
- Once the target sequence is located, various conformational changes occur in the enzyme as well as the DNA. These conformational changes, in turn, activate catalytic center.
- The phosphodiester bond is hydrolyzed, and the product is released.
C. Type III Restriction Enzyme
- 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.
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.
Cleavage patterns of HindIII, SmaI, EcoRI, and BamHI are described as below. Most of the enzymes recognize sequences which are 4 to 6 base pairs long. However, they can also be up to 8 base pairs in length.
The process of cleavage of DNA by the restriction enzyme culminates with the formation of either sticky ends or blunt ends.
The blunt-ended fragments can be joined with the DNA fragment only with the aid of linkers and adapters.
- 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.