More than 30 years ago, when people were studying the host-specific restriction-modification phenomenon of bacteriophages, restriction endonucleases were discovered for the first time. Bacteria can resist the invasion of new viruses, and this method of limiting the survival of viruses can be attributed to the restriction enzymes inside the cells that can destroy foreign DNA. The first restriction enzymes discovered include EcoR I and EcoR II from Escherichia coli, and Hind II and Hind III from Heamophilus influenzae. These enzymes can cut DNA at specific sites to produce gene fragments that can be ligated in vitro. Researchers soon discovered that endonuclease is a very useful tool for studying gene composition, function and expression.
Restriction endonucleases come in various forms. In terms of size, they can be as small as Pvu II (157 amino acids) or larger than Cje I of 1250 amino acids. Among 3000 restriction endonucleases that have been purified and classified, more than 250 specific recognition sequences have been found. While analyzing the biochemical aspects of cell extracts, people also use computers to analyze known genomic data, hoping to make more discoveries. Although the recognition sequences of many newly discovered enzymes are duplicated with existing ones, there are still enzymes that recognize new sites constantly being discovered.
The main function of restriction enzymes is to protect bacteria from phage infection, and they perform their functions as part of the microbial immune mechanism. When a bacteria without restriction endonuclease is infected by a virus, most of the virus particles can be successfully infected. The greater the number of restriction endonucleases, the more protective effects will be achieved, and one having 4 to 5 independent restriction enzymes will make the cell indestructible.
Traditionally, restriction endonucleases are divided into three categories according to subunit composition, restriction position, recognition site, and cofactors. However, the results of protein sequencing show that there are many variations of restriction enzymes, and if they are classified at the molecular level, there should be far more than these three.
Type I restriction endonucleases are a class of protein complexes with multiple subunits that have both restriction endonuclease and modification enzyme activities. They cut DNA strands arbitrarily far away from the recognition site. In the past, it was thought that type I restriction endonucleases were rare, but now the results of genome sequencing have found that this type of enzyme is actually very common. Although type I enzymes are of great significance in biochemical research, they are not practical because they do not produce definite restriction fragments.
Type II enzymes specifically cut DNA strands at defined sites in or near their recognition sites. They produce defined restriction fragments, so they are the only one used for DNA analysis and cloning among the three types of restriction endonucleases. Type II restriction endonucleases are composed of a group of proteins with different traits and sources. Therefore, the amino acid sequence of any restriction endonuclease may be completely different from the amino acid sequence of another restriction endonuclease. In fact, it is speculated from the known situation that these enzymes are likely to be produced independently during the evolution process, rather than from the same ancestor.
The most common type II restriction endonucleases are enzymes that cut in the recognition sequence such as Hha I, Hind III and Not I. Such enzymes constitute the main part of commercial enzymes. Most of these enzymes bind to DNA in the form of homodimers, thus recognizing symmetric sequences; but very few enzymes bind to DNA as monomers and recognize asymmetric sequences. Some enzymes recognize contiguous sequences (e.g. EcoRI recognizes GAATTC); while others recognize discrete sequences (e.g. Bgl I recognizes GCCNNNNNGGC). Restriction endonuclease cleavage produces a 3'hydroxyl end and a 5'phosphate group. Their activity requires magnesium ions, and the corresponding modified enzymes require the presence of S-methionine adenosine. These enzymes are generally relatively small, and the subunits are generally around 200-300 amino acids.
Type III restriction endonucleases are also enzymes with both restriction and modification functions. They cut the DNA strand outside the recognition site and require the recognition site to be an inverted repeat sequence. They rarely produce completely cut fragments, so they have no practical value.
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