Restrictase - what is it? Definition and classification of restriction enzymes

Restriction enzymes are special enzymes that can cut DNA chains in strictly defined places. The specificity of the action is the main difference between this group of proteins and other types of endonucleases. Due to this unique property, restriction enzymes are widely used in genetic engineering.

Restriction enzymes are unique bacterial enzymes. For the first time, these proteins were isolated in 1968 from strains of Esherihia colli. Further studies revealed the presence of restriction endonucleases in the unicellular alga Chlorella, as well as in several cultures of the yeast Sachoromyces and Pichia. In the cells of other eukaryotes, restriction enzymes have not yet been detected.

general characteristics

Restriction enzymes are enzymes belonging to the hydrolase group, since they catalyze the hydrolysis of the phosphodiester linkage between nucleotides in the DNA chain. The place where the cutting occurs is called a restriction site. The latter can be located both inside the recognized sequence and at a distance from it.

A restriction-specific recognition site usually consists of 4-8 nucleotides. Since the nitrogenous bases are turned into a double helix, the enzyme finds the desired sequence through interaction with a large groove of DNA.

Each restrictase has its own recognition site and can only function after binding to it.

Biological role

Restriction enzyme bacteria are an essential tool to protect cells from bacteriophages. The main task of the enzymes of this group is to cut the foreign genetic material that has penetrated the cell. Fragmentation of a nucleic acid prevents its replication, and hence the reproduction of the virus.

The sequences to which restriction enzymes are specific occur with a certain periodicity in the genome of the bacterium itself and therefore undergo methylation, which does not allow restriction enzymes to recognize their targets. This process is carried out by enzymes methylases.

The mechanism aimed at cutting foreign DNA and protecting its own from nuclease activity is called a restriction-modification system. Some types of restriction enzymes are capable of both endonuclease and methylase activity.

Currently, more than 1000 bacterial restriction enzymes are known. Very often they are considered not as separate enzymes, but as a complex of restriction-modification systems.

Features of the action of restriction enzymes

After binding to the recognition sequence of the restriction endonuclease, the DNA strands are sequentially cleaved. In this case, two variants of the ends can form:

• Sticky - single-stranded sequences complementary to each other, which can easily "cross-link" back. They are formed during a stepped cutting path, when the split points of the two chains do not coincide;
• Dull - single-stranded regions at the site of DNA rupture are not formed, the points of chain cutting are opposite each other.

For the implementation of endonuclease activity, restriction enzymes require a cofactor in the form of Mg ²⁺ . Located at the gap, this ion interferes with the configuration of DNA, which contributes to subsequent cleavage.

Restriction Classification

According to the structure and mechanism of action, all restriction enzymes are divided into 2 classes. The first includes enzymes with exclusively restriction activity, and methylation is carried out by another protein with a similar substrate specificity.

Class II restriction enzymes are multienzyme complexes capable of both cutting and modifying DNA. These proteins are composed of 3 functional subunits:

• site recognition;
• methylating;
• restrictive.

There is another variety of restriction enzymes of the same class, which is characterized by a structure of two subunits. Such enzymes are called bifunctional.

Methylase and endonuclease activity in different restriction enzymes can be carried out simultaneously or exclude each other. The effect of such enzymes depends on the reaction conditions.

Within two classes, three types of restriction enzymes (I, II, and III) will be distinguished, which differ in the following characteristics:

• structural organization;
• functions performed;
• features of substrate specificity;
• need for cofactors;
• the location of the restriction site relative to the recognition site;
• the reversibility of the restriction hydrolysis reaction;
• ATP energy requirement;
• mechanism of action.

Type II corresponds to the first class of restriction endonucleases, and the first and third types are varieties of class II.

In addition to the three main types of restriction enzymes, two more types are distinguished:

• IIS - differs from type II in that the restriction-modification system is encoded not by two, but by three genes, and the cutting site is outside the recognition sequence (at a fixed distance);
• Mcr / Mrr - contains several different subunits in the absence of modification activity, GTP is required for work, breaks down DNA with methylated adenine and cytosine.

The restriction enzymes of the first and third types are characterized by incomplete hydrolysis of DNA chains and therefore are practically not used in genetic engineering technologies.

Nomenclature

Usually, the name of the restrictase contains information about which microorganism it was isolated from. In this case, the information is indicated in the following order:

• genus and species;
• strain;
• enzyme number (if this microorganism contains several restriction-modification systems).

Sometimes the name includes information on the localization of genes encoding a restrictase.

The nomenclature for restriction enzymes was developed by Smith and Nathans. It is based on the following principles:

• The first Latin letter denotes the genus, and the next two letters indicate the type of microorganism.
• Next, beech latin indicates a strain or extrachromosomal element encoding a restrictase.
• At the end, the Roman number indicates the number of the enzyme.

Thus, the second restriction enzyme isolated from strain d of the bacterium Haemophilus influenzae will be designated HindII.

Isoshizomers

There is another classification of restriction enzymes based on their substrate specificity.

Isoshizomers are restriction enzymes with different origins, but the same substrate specificity. Such restriction enzymes can be isolated even from distant bacterial taxa. The discovery of isoshizomers refuted the assumption of strict taxon-specificity of restrictase enzymes, which claimed that the recognition sites for these enzymes in different types of microorganisms are unique.

Neoshizomer

The restriction enzymes having a common recognition site, but different restriction sites, are called neoshizomers. For example, the sequence CCCGGG is specific for SmaI and XmaI enzymes, but in one case, the cut is in the middle (CCC / GGG), and in the other, the bond between the first and second nucleotides (C / CCGGG) is cleaved.

Isocaudomers

Isocaudomers include restriction enzymes with different substrate specificity, which form the same ends. It is assumed that the proteins of this group are not related.

The use of restriction enzymes

Restriction enzymes are one of the main molecular tools used in recombinant DNA technology. Thanks to these enzymes, it became possible to cut out certain fragments or genes from the genomes of various organisms, and then crosslink them using ligases.

Particularly convenient is the use in genetic engineering of restriction enzymes that form the same sticky ends. Such DNA fragments are easily linked according to the principle of complementarity.