Restriction enzyme

Restriction enzymes, also known as restriction endonucleases, are specialised proteins that cleave DNA at or near specific nucleotide sequences known as restriction sites. These enzymes play a fundamental role in the natural defence systems of bacteria and archaea, where they protect the host cell from invading viral DNA. Their precision in recognising and cutting DNA has made them indispensable tools in molecular biology, biotechnology, and genetic engineering.
Restriction enzymes belong to a broader family of endonucleases, but are distinguished by their strict sequence specificity. Thousands of such enzymes have been identified and characterised, offering a wide array of recognition sites and cleavage patterns for laboratory use.

Discovery and Early Research

The concept of restriction enzymes emerged from studies on bacteriophages, particularly lambda phage, during the early 1950s. Researchers observed that bacteriophages reproduced efficiently in certain strains of Escherichia coli but poorly in others, despite being genetically identical. This phenomenon, termed “host-controlled restriction and modification”, suggested that bacterial hosts possessed mechanisms for degrading foreign DNA while protecting their own.
Work in the 1960s by Werner Arber and Matthew Meselson demonstrated that the restriction effect was due to the enzymatic cleavage of phage DNA. These enzymes were accordingly named restriction enzymes. The first restriction enzymes described were Type I enzymes, which cut DNA at sites distant from their recognition sequences.
A major breakthrough occurred in 1970 when Hamilton O. Smith and colleagues isolated and characterised HindII, the first Type II restriction enzyme, from Haemophilus influenzae. Unlike Type I enzymes, Type II enzymes cut DNA precisely at defined sites, making them ideal for laboratory applications. Their discovery transformed genetic research.
Daniel Nathans and Kathleen Danna subsequently demonstrated that restriction enzymes could be used to generate reproducible DNA fragments and to map viral genomes using polyacrylamide gel electrophoresis. In recognition of their contributions, Arber, Nathans, and Smith received the 1978 Nobel Prize in Physiology or Medicine.

Biological Function and Restriction–Modification Systems

In prokaryotes, restriction enzymes function as part of a restriction–modification (R–M) system. While restriction enzymes cleave foreign DNA, the host genome is protected by methyltransferases that add methyl groups to specific bases within the same recognition sites. This methylation prevents self-DNA from being targeted and ensures selective cleavage of invading genetic material.
More than 3,600 restriction endonucleases with over 250 specificities have been documented. Over 800 of them are commercially available and extensively used in cloning, genome mapping, and recombinant DNA technology.

Recognition Sites and Sequence Properties

Restriction enzymes identify specific sequences of nucleotides, generally between four and eight base pairs in length. The length of the recognition site determines its frequency within a genome: shorter sites occur more frequently, while longer sites are rarer.
Many recognition sequences are palindromic, meaning that the sequence reads the same forwards and backwards on complementary strands. Two major forms of palindromes exist:

• Mirror-like palindromes, which read identically on a single strand.
• Inverted repeat palindromes, where each strand contains complementary sequences that form a symmetrical motif when read in opposite directions.

Inverted repeats are the most common and biologically significant form, serving as the basis for cleavage by many restriction enzymes.
Cleavage generates different types of DNA ends:

• Sticky ends, with short single-stranded overhangs that can form hydrogen bonds with complementary sequences.
• Blunt ends, where both DNA strands are cut at the same position.

Restriction enzymes that recognise the same sequence are known as isoschizomers, whereas those that recognise the same site but cut at different positions are termed neoschizomers.

Classification of Restriction Enzymes

Restriction enzymes are traditionally divided into five types based on structure, cofactor requirements, recognition site characteristics, and cleavage positions.
Type I enzymesThese were the first to be discovered. They recognise asymmetrical sequences composed of two specific regions separated by a variable spacer. These enzymes cut DNA at unpredictable distances—often over a thousand base pairs away from the recognition site. Their activity requires ATP, S-adenosyl methionine, and magnesium ions. They are multifunctional proteins containing three subunits:

• HsdR, responsible for restriction activity
• HsdM, involved in DNA methylation
• HsdS, determining sequence specificity

Type I enzymes also function as molecular motors, translocating DNA until cleavage occurs.
Type II enzymesType II enzymes recognise palindromic sites and cleave within or adjacent to those sequences. They generally require only magnesium ions and are the most widely used in molecular biology due to their precision. They operate independently of methylation processes and cleave DNA predictably, making them ideal for cloning and recombinant DNA applications.
Type III enzymesThese enzymes cut DNA a short distance from their recognition sites. Their activity requires ATP, but they do not hydrolyse it. S-adenosyl L-methionine enhances activity but is not essential. They exist as complexes that include a modification methylase.
Type IV enzymesThese enzymes target modified DNA, including methylated and hydroxymethylated sequences, often found in bacteriophage genomes.
Type V enzymesType V enzymes are guided by RNA molecules, most notably represented by CRISPR–Cas systems. These enzymes recognise sequences through RNA–DNA base pairing rather than intrinsic protein–DNA interactions.

Evolutionary Origins

Restriction enzymes appear to have evolved from an ancestral gene and became widespread in bacteria and archaea through horizontal gene transfer. Some evidence suggests that they originated as self-propagating genetic elements, later co-opted into host defence mechanisms.

Applications in Molecular Biology

The development of restriction enzymes made modern genetic engineering possible. Their ability to produce predictable DNA fragments laid the foundation for recombinant DNA technology. Among the many applications are:

• Construction of recombinant plasmids
• Gene cloning
• DNA sequencing and mapping
• Production of therapeutic proteins such as human insulin
• Diagnostic techniques in genomics and forensic science