Basics of Restriction Endonucleases

Today about 4,000 restriction enzymes have been characterized, and over 600 of those are commercially available. REBASE is a useful, browsable resource for comprehensive and up-to-date information about restriction enzymes, including specificity, sensitivity, and commercial sources [1].

In the early 1950s, a number of research teams observed differences in the efficiency of bacteriophage infection on different bacterial host strains of the same species [2,3]. This was described by Grasso and Paigen: When phage λ propagated in one strain of bacteria (e.g., E. coli C) was used to infect another strain of the same species of bacteria (e.g., E. coli K), a marked decrease in the rate of infection was noted compared to re-infection of the host strain (E. coli C). The new host (E. coli K) seemed to select against or “restrict” the incoming phage. The researchers also noted this was not a hereditary phenomenon, because the phage that did grow on the new strain could infect that strain at more typical rates after one round of infection. The observed phenomenon was defined as “host control variation” and became an area of intense research to discover the underlying mechanisms [4].

Figure 2. Landmarks in restriction endonucleases development.

It was not until the 1960s that mechanisms underlying host control variation were determined to involve enzymatic cleavage of the phage DNA, which led to the discovery and isolation of restriction enzymes. In the early 1960s, Werner Arber observed that the host-range determinant resided on the phage DNA, and subsequent experiments showed that methionine was involved in host protection [5]. These findings ultimately led to the proposal of a restriction-modification (R-M) system, in which a restriction enzyme and a methylase from the host work together to cleave foreign viral (non-methylated) DNA while keeping the host DNA protected through methylation [6].

Interestingly, most of the early work on R-M systems was on Type I and III groups of restriction enzymes, classified based on aspects of their structure and function. The full potential of restriction enzymes did not become apparent until Kent Wilcox and Hamilton Smith discovered HindII (HincII), the first restriction enzyme of the Type II class [7]. HindII recognizes a specific symmetrical DNA sequence and cleaves in a defined manner within that recognition sequence. This feature, found in most early Type II restriction enzymes, led Kathleen Danna and Daniel Nathans to use HindII in the physical mapping of simian virus 40 DNA [8], a process known as restriction enzyme mapping.

For their pioneering work with restriction enzymes, Daniel Nathans, Hamilton Smith, and Werner Arber were awarded the 1978 Nobel Prize in Physiology or Medicine.

Learn more about Thermo Scientific FastDigest HincII restriction enzyme

With the discovery of DNA ligase, in combination with the growing family of site-specific cutting restriction enzymes, recombinant DNA technology was born.

Read more on recombinant DNA technology

Find out more about Thermo Scientific T4 DNA Ligase

Restriction endonucleases occur naturally in prokaryotic and eukaryotic systems. When occurring in their cognate environment, such as a bacterial host, they perform key function as a defensive mechanism as part of restriction-modification (R-M) system. In this system, the host cell produces both a restriction enzyme and a corresponding DNA methyltransferase. The methyltransferase modifies the host's own DNA by adding methyl groups to specific bases within the recognition sequences, protecting it from cleavage by the restriction enzyme. Thus, the restriction enzyme only cleaves unmethylated (foreign) DNA, while the host DNA remains protected due to methylation. This selective cleavage ensures that only foreign DNA is targeted and degraded.

In some cases, restriction-modification systems can influence gene expression and regulation within the host. The methylation status of DNA can affect the binding of transcription factors and other regulatory proteins, thereby modulating gene activity.

Furthermore, restriction endonucleases can facilitate genetic recombination and genome rearrangement by cutting DNA at specific sites. This can lead to the exchange of genetic material between different DNA molecules, contributing to genetic diversity and evolution.

The naming convention takes into account three characteristics of the enzyme’s organismal origin—genus, species, and strain or serotype—to develop a shortened name followed by roman numerals to represent multiple restriction enzymes from the same strain [9]. For example, the enzyme HincII (or Hind III in earlier nomenclature) represents:

• “H” for Haemophilus
• “in” fo influenzae
• “d” for serotype d
• “III” to distinguish from other restriction enzymes from Haemophilus influenza serotype d, for example, HindII (HincII) vs. HindIII

Restriction enzymes are categorized into four classes, based on their structural complexity, recognition sequence, cleavage site position, and cofactor requirement. Table 1 summarizes the distinguishing characteristics of these classes.

Table 1. Restriction enzyme classes and characteristics.

Type II restriction endonucleases are the most commonly used restriction enzymes in several research applications such as cloning, forensic DNA analysis, and molecular taxonomy.

In cloning, restriction enzymes enable precise DNA manipulation by cutting DNA at specific sequences, allowing researchers to isolate and insert genes into vectors for replication and expression in host cells. Ligase, which enzymatically joins 5′ phosphates and 3′ hydroxyls at DNA termini, enables rearrangement and connecting of DNA molecules with 5′-phosphate and 3′-hydroxyl termini generated by restriction enzymes. This combination of DNA digestion with restriction enzymes and ligation is the fundamental principle of recombinant DNA cloning technology.

The specific cutting pattern of these enzymes led to their use in restriction fragment length polymorphism (RFLP) analysis, which is a basis of forensic studies and molecular taxonomy. Genetic variations among species or DNA samples can be compared by DNA fragment patterns generated by enzyme digestion.

Due to their usefulness in molecular biology research, the type II restriction enzymes are the most studied class of enzymes and comprise the largest group. Over 3,500 Type II restriction enzymes have been characterized and subcategorized further into groups such as Type IIP, IIA, IIB, IIC, IIS, etc., where Type IIP enzymes, which recognize palindromic (symmetric) target sequences, are the most prevalent among commercially available restriction enzymes [10].

Learn more about type IIs cloning

For a deeper dive into how Type IIS restriction enzymes are used in cloning applications, check out our blog post Restriction Endonuclease 101: From Basics to Golden Gate Cloning.

Another important way to classify and compare restriction enzymes is isoschizomers and neoschizomers.

• Isoschizomers are restriction enzymes that have the same recognition sequence and the same specificity. For instance, Thermo Scientific BshT1 and its isoschizomer Agel recognize and cleave 5′-A↓CCGGT-3′ in the same pattern. Nevertheless, a set of isoschizomers may differ in site preferences, reaction conditions, methylation sensitivity, and star activity.
• Neoschizomers recognize the same nucleotide sequence but cleave DNA at different positions. Examples of neoschizomers are Thermo Scientific FastDigest SmaI (5′-CCC↓GGG-3′) and Thermo Scientific Cfr9I (XmaI) (5′-C↓CCGGG-3′), which both recognize 5′-CCCGGG-3′ but cleave them differently and thus generate different types of ends (in this case, blunt ends for SmaI and 5′ protruding ends for XmaI).

Read more on isoschizomers and neoschizomers to target methylated DNA

The availability of different specificities in both recognition sequence and cleavage pattern has made restriction enzymes an extremely flexible and powerful set of tools for characterizing and manipulating genetic material.

1. Roberts RJ, Vincze T, Posfai J, et al. (2015) REBASE—a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res 43:D298–D299. doi: 10.1093/nar/gku1046.
2. Luria SE, Human ML (1952) A nonhereditary, host-induced variation of bacterial viruses. J Bacteriol 64:557–569. doi: 10.1128/jb.64.4.557-569.1952.
3. Bertani G, Weigl JJ (1953) Host controlled variation in bacterial viruses. J Bacteriol 65:112–121. doi: 10.1128/jb.65.2.113-121.1953.
4. Grasso RJ, Paigen K (1968) Loss of host-controlled restriction of λ bacteriophage in Escherichia coli following methionine deprivation. J Virol 2:1368–1373. doi: 10.1128/JVI.2.12.1368-1373.1968.
5. Arber W (1965) Host specificity of DNA produced by Escherichia coli V. The role of methionine in the production of host specificity. J Mol Biol 11:247–256. doi: 10.1016/s0022-2836(65)80055-9.
6. Arber W (1965) Host-controlled modification of bacteriophage. Annu Rev Microbiol 19:365–378. doi: 10.1146/annurev.mi.19.100165.002053.
7. Kelly TJ, Smith HO (1970) A restriction enzyme from Hemophilus influenzae II. J Mol Biol 51:393–409. doi: 10.1016/0022-2836(70)90150-6.
8. Danna K, Nathans D (1971) Specific cleavage of simian virus 40 DNA by restriction endonuclease from Hemophilus influenza. Proc Natl Acad Sci USA 68:2913–2917. doi: 10.1073/pnas.68.12.2913.
9. Smith, HO, Nathans D (1973) Letter: a suggested nomenclature for bacterial host modification and restriction systems and their enzymes. J Mol Biol 81:419–423. doi: 10.1016/0022-2836(73)90152-6.
10. Pingoud A, Wilson GG, Wende W (2014) Type II Restriction Endonucleases – a Historical Perspective and More. Nucleic Acids Res 42:7489–7527. doi: 10.1093/nar/gku447.