RESTRICTION ENZYMES: FUNCTIONS, TYPES, AND EXAMPLES - BIOLOGY - 2025

The restriction enzymes are endonucleases employed by certain archaea and bacteria to inhibit or "restrict" the spread of viruses inside. They are especially common in bacteria and are part of their defense system against foreign DNA known as the restriction / modification system.

These enzymes catalyze the cleavage of double-band DNA at specific locations, reproducibly and without the use of additional energy. Most require the presence of cofactors such as magnesium or other divalent cations, although some also require ATP or S-adenosyl methionine.

HindIII restriction enzyme reaction scheme (Source: Helixitta via Wikimedia Commons)

Restriction endonucleases were discovered in 1978 by Daniel Nathans, Arber Werner, and Hamilton Smith, who received the Nobel Prize in medicine for their discovery. Their name generally derives from the organism where they are first observed.

Such enzymes are widely used in the development of DNA cloning methods and other molecular biology and genetic engineering strategies. Their specific sequence recognition characteristics and the ability to cut sequences close to recognition sites make them powerful tools in genetic experimentation.

Fragments generated by restriction enzymes that have acted on a particular DNA molecule can be used to recreate a "map" of the original molecule by using information about the sites where the enzyme cut DNA.

Some restriction enzymes may have the same recognition site on DNA, but they do not necessarily cut it in the same way. Thus, there are enzymes that cut leaving blunt ends and enzymes that cut leaving cohesive ends, which have different applications in molecular biology.

Currently there are hundreds of different commercially available restriction enzymes, offered by different commercial houses; These enzymes function as "custom" molecular scissors for different purposes.

Features

Restriction enzymes fulfill the opposite function of polymerases, since they hydrolyze or break the ester bond within the phosphodiester bond between adjacent nucleotides in a nucleotide chain.

In molecular biology and genetic engineering they are widely used tools for the construction of expression and cloning vectors, as well as for the identification of specific sequences. They are also useful for the construction of recombinant genomes and have great biotechnological potential.

Recent advances in gene therapy make current use of restriction enzymes for the introduction of particular genes into vectors that are vehicles for the transport of such genes into living cells, and that probably have the ability to insert into the cellular genome to perform permanent changes.

Mechanism of action

Restriction enzymes can catalyze double-band DNA cleavage, although some are capable of recognizing single-band DNA sequences and even RNA. Cutting occurs after recognition of the sequences.

The mechanism of action consists of the hydrolysis of the phosphodiester bond between a phosphate group and a deoxyribose in the skeleton of each DNA strand. Many of the enzymes are capable of cutting at the same site that they recognize, while others cut between 5 and 9 base pairs before or after it.

Normally these enzymes cut at the 5 'end of the phosphate group, giving rise to DNA fragments with a 5' phosphoryl end and a 3 'terminal hydroxyl end.

Since proteins do not come in direct contact with the recognition site in DNA, they must be translocated successively until the specific site is achieved, perhaps by means of "sliding" mechanisms on the DNA strand.

During enzymatic cleavage, the phosphodiester bond of each of the DNA strands is positioned within one of the active sites of restriction enzymes. When the enzyme leaves the recognition and cleavage site, it does so through non-specific transient associations.

Types

Five types of restriction enzymes are currently known. Here is a brief description of each one:

Type I restriction enzymes

These enzymes are large pentameric proteins with three subunits, one for restriction, one for methylation, and one for sequence recognition in DNA. These endonucleases are multifunctional proteins capable of catalyzing restriction and modification reactions, they have ATPase activity and also DNA topoisomerase.

Enzymes of this type were the first endonucleases to be discovered, they were first purified in the 1960s and have been studied in great depth ever since.

Type I enzymes are not widely used as a biotechnological tool, since the cleavage site can be at a variable distance of up to 1,000 base pairs from the recognition site, which makes them unreliable in terms of experimental reproducibility.

Type II restriction enzymes

They are enzymes composed of homodimers or tetramers that cut DNA at defined sites between 4 and 8 bp in length. These cleavage sites are typically palindromic, that is, they recognize sequences that are read in the same way in both directions.

Many of the type II restriction enzymes in bacteria cut DNA when they recognize its foreign character, since it does not have the typical modifications that its own DNA should have.

These are the simplest restriction enzymes since they do not require any cofactor other than magnesium (Mg +) to recognize and cut DNA sequences.

The precision of type II restriction enzymes in recognizing and cutting simple sequences in DNA at precise positions makes them one of the most widely used and indispensable in most branches of molecular biology.

Within the group of type II restriction enzymes there are multiple subclasses classified according to certain properties that are unique to each one. The classification of these enzymes is done by adding letters of the alphabet, from A to Z following the name of the enzyme.

Some of the subclasses best known for their usefulness are:

Subclass IIA

They are dimers of different subunits. They recognize asymmetric sequences and are used as ideal precursors for the generation of cutting enzymes.

Subclass IIB

They are made up of one or more dimers and cut DNA on both sides of the recognition sequence. They cut both strands of DNA a base pair interval ahead of the recognition site.

Subclass IIC

Enzymes of this type are polypeptides with functions of division and modification of DNA strands. These enzymes cut both strands asymmetrically.

Subclass IIE

The enzymes of this subclass are the most used in genetic engineering. They have a catalytic site and generally require an allosteric effector. These enzymes need to interact with two copies of their recognition sequence to perform efficient cleavage. Within this subclass are the enzymes EcoRII and EcoRI.

Type III restriction enzymes

Type III restriction endonucleases are composed of only two subunits, one is responsible for DNA recognition and modification, while the other is responsible for sequence cleavage.

These enzymes require two cofactors for their function: ATP and magnesium. Restriction enzymes of this type possess two asymmetric recognition sites, translocate DNA in an ATP-dependent manner and cut it between 20-30 bp adjacent to the recognition site.

Type IV restriction enzymes

Type IV enzymes are easy to identify as they cut DNA with methylation marks, they are made up of several different subunits that are responsible for recognizing and cutting the DNA sequence. These enzymes use GTP and divalent magnesium as cofactors.

Specific cleavage sites include nucleotide strands with methylated or hydroxymethylated cytosine residues on one or both strands of nucleic acids.

Type V restriction enzymes

This classification groups the CRISPER-Cas type enzymes, which identify and cut specific DNA sequences from invading organisms. Cas enzymes use a strand of CRISPER synthesized guide RNA to recognize and attack invading organisms.

Enzymes classified as type V are polypeptides structured by type I, II and II enzymes. They can cut sections of the DNA of almost any organism and with a wide range of length. Their flexibility and ease of use make these enzymes one of the most widely used tools in genetic engineering today, along with type II enzymes.

Examples

Restriction enzymes have been used for the detection of DNA polymorphisms, especially in population genetic studies and evolutionary studies using mitochondrial DNA, in order to obtain information about the rates of nucleotide substitutions.

Currently, the vectors used for the transformation of bacteria for various purposes possess multicloning sites where recognition sites for multiple restriction enzymes are found.

Among these enzymes the most popular are EcoRI, II, III, IV and V, obtained and described for the first time from E. coli; HindIII from H. influenzae and BamHI from B. amyloliquefaciens.

References

1. Bickle, TA, & Kruger, DH (1993). Biology of DNA Restriction. Microbiological Reviews, 57 (2), 434–450.
2. Boyaval, P., Moineau, S., Romero, DA, & Horvath, P. (2007). CRISPR Provides Acquired resistance against viruses in prokaryotes. Science, 315 (March), 1709–1713.
3. Goodsell, D. (2002). The molecular perspective: Restriction Endonucleases. Stem Cells Fundamentals of Cancer Medicine, 20, 190–191.
4. Halford, SE (2001). Hopping, jumping and looping by restriction enzymes. Biochemical Society Transactions, 29, 363-373.
5. Jeltsch, A. (2003). Maintenance of species identity and controlling speciation of bacteria: a new function for restriction / modification systems? Gene, 317, 13-16.
6. Krebs, J., Goldstein, E., & Kilpatrick, S. (2018). Lewin's Genes XII (12 ed.). Burlington, Massachusetts: Jones & Bartlett Learning.
7. Li, Y., Pan, S., Zhang, Y., Ren, M., Feng, M., Peng, N.,… She, Q. (2015). Harnessing Type I and Type III CRISPR-Cas systems for genome editing. Nucleic Acids Research, 1–12.
8. Loenen, WAM, Dryden, DTF, Raleigh, EA, & Wilson, GG (2013). Type I restriction enzymes and their relatives. Nucleic Acids Research, 1–25.
9. Nathans, D., & Smith, HO (1975). Restriction Endonucleases in the analysis and restructuring of DNA molecules. Annu. Rev. Biochem., 273–293.
10. Nei, M., & Tajima, F. (1981). Dna polymorphism detectable by restriction endonucleases. Genetics, 145-163.
11. Pingoud, A., Fuxreiter, M., Pingoud, V., & Wende, W. (2005). Cellular and Molecular Life Sciences Type II restriction endonucleases: structure and mechanism. CMLS Cellular and Molecular Life Sciences, 62, 685–707.
12. Roberts, R. (2005). How restriction enzymes became the workhorses of molecular biology. PNAS, 102 (17), 5905–5908.
13. Roberts, RJ, & Murray, K. (1976). Restriction endonucleases. Critical Reviews in Biochemistry, (November), 123-164.
14. Stoddard, BL (2005). Homing endonuclease structure and function. Quarterly Reviews of Biophysics, 1–47.
15. Tock, MR, & Dryden, DTF (2005). The biology of restriction and anti-restriction. Current Opinion in Microbiology, 8, 466–472.
16. Wilson, GG, & Murray, NE (1991). Restriction and Modification Systems. Annu. Rev. Genet., 25, 585-627.
17. Wu, Z., & Mou, K. (2016). Genomic insights into Campylobacter jejuni virulence and population genetics. Infec. Dis. Transl. Med., 2 (3), 109–119.
18. Yuan, R. (1981). Structure and Mechanism of Multifunctional Restriction Endonucleases. Annu. Rev. Biochem., 50, 285-315.