Restriction Enzyme: Definition, Types & Usages

What is a Restriction Enzyme Simple Definition?

Restricted endonucleases or restriction nucleases are enzymes that cut DNA into fragments at specific recognition sites or nearby (referred to as restriction sites) within the molecule. Restriction endonucleases are widely distributed, with at least one type found in almost every bacterial genus and species, with some genera harboring dozens of different types. For example, within the genus Haemophilus, 22 types have been discovered. Some bacterial strains have very low enzyme levels, making qualitative separation difficult. However, in certain strains, enzyme levels can be extremely high, such as in E. coli strain pMB4 (EcoRI enzyme) and H. aegyptius (enough enzyme to digest 10g of λ phage DNA). Bacteria are the primary source of restriction endonucleases, especially highly specific Type I restriction endonucleases.

Laboratory manipulation of restriction endonucleases.Laboratory manipulation of restriction endonucleases.

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Types of Restriction Enzyme

Basis of Classification of Restriction Enzymes

Restriction enzymes are broadly categorized into four primary types—Type I, II, III, and IV—based on the R-M system composition, DNA recognition sequences, cleavage sites, and auxiliary factors.

What are the 4 Types of Restriction Enzymes?

  • Type I

The first discovered restriction enzymes, EcoKI and EcoBI, are typical Type I restriction enzymes. The common characteristics of such restriction enzymes are:

(1) They are composed of a pentameric complex (2R+2M+S) consisting of restriction enzyme (R, encoded by the hsdR gene), methylase (M, encoded by the hsdM gene), and DNA-specific sequence recognition (S, encoded by the hsdS gene), with a molecular weight reaching 400 kDa. They possess both restriction and methylation activities.

(2) Cleavage typically occurs at positions far from the recognition site (sometimes even more than 2 kb apart), and the distance is variable.

(3) They require ATP, Mg2+, and S-adenosyl methionine (SAM) as cofactors.

From the second characteristic, it is evident that Type I restriction enzymes are unable to generate defined cleavage products, making them unsuitable as tools for genetic engineering. Common genetic engineering strains typically require mutation or inactivation of the hsd genes encoding Type I restriction enzymes to prevent degradation of exogenous plasmids.

  • Type II

Hamilton Smith first discovered HindII from Haemophilus influenzae, while Dan Nathans used HindII and other restriction enzymes to cleave SV40 virus DNA, thus constructing the first restriction physical map of a genome. HindII here is the first Type II restriction enzyme discovered. Of the restriction enzymes identified to date, over 95% belong to Type II. The common characteristics of this type of restriction enzyme are:

(1) Most Type II restriction enzymes and their corresponding methylases are independent proteins, although there are also a few fusion proteins of restriction enzymes and methylases.

(2) Cleavage sites are located either within the recognition sequence or at a fixed distance from the recognition site.

(3) Most Type II restriction enzymes require only Mg2+ as a cofactor.

Type II restriction enzymes can generate accurately controllable enzyme cleavage products, making them easy to identify and recombinantly express, and thus have become the primary restriction enzymes used in genetic engineering.

  • Type III

Type III R-M systems were first discovered during the study of λ phage infection of P1 lysogenic Escherichia coli. Similar R-M systems have since been found in other bacteriophage-bacteria interactions. Type III restriction enzymes often function as homodimers, with each monomeric protein containing both restriction and methylation domains. They require Mg2+, ATP, and SAM as cofactors. The recognition sites of Type III restriction enzymes consist of two inverted sequences on the same DNA molecule, with most containing an A-rich sequence. While individual recognition sequences are typically non-palindromic, the two recognition sequences are reverse complementary. The spacer sequences between the two recognition sites lack specificity and vary greatly in length. The cleavage sites of Type III restriction enzymes are located at a fixed distance from one of the recognition sequences, and sometimes the cleavage is incomplete, making them rarely used as tool enzymes.

  • Type IV

Type IV restriction enzymes, initially discovered during the T4 bacteriophage and Escherichia coli interaction, demonstrate diverse compositions and can recognize various methylation sites like m6A, m5C, and hm5C. Despite their low sequence specificity, enzymes like McrA, McrBC, and Mrr from E. coli are notable examples. In some genetically modified strains, it might be necessary to mutate or knock out genes encoding Type IV restriction enzymes to avoid interfering with foreign gene expression. Recent advances in epigenetics have revealed that these enzymes can discern different methylation modifications. Thus, researchers are exploring their use in epigenetic studies. For example, McrBC has been used to analyze CpG dinucleotide methylation, offering insights into epigenetic regulation mechanisms. This application highlights the growing importance of Type IV restriction enzymes in understanding epigenetic phenomena and their biological implications.

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Common Restriction Endonucleases

Note: The * stands for blunt ends.

NameSourceIdentification/Cutting Sites
EcoRIEscherichia coli5'---G AATTC---3'
3'---CTTAA G---5'
BamHIBacillus amyloliquefaciens5'---G GATCC---3'
3'---CCTAG G---5'
HindIIIHaemophilus influenzae5'---A AGCTT---3'
3'---TTCGA A---5'
TaqIThermus aquaticus5'---T CGA---3'
3'---AGC T---5'
NotINocardia otitidis5'---GC GGCCGC---3'
3'---CGCCGG CG---5'
HinfIHaemophilus influenzae5'---G ANTC---3'
3'---CTNA G---5'
Sau3AStaphylococcus aureus5'--- GATC---3'
3'---CTAG ---3'
PovII*Proteus vulgaris5'---CAG CTG---3'
3'---GTC GAC---5'
SmaI*Serratia marcescens5'---CCC GGG---3'
3'---GGG CCC---5'
HaeIII*Haemophilus egytius5'---GG CC---3'
3'---CC GG---5'
AluI*Arthrobacter luteus5'---AG CT---3'
3'---TC GA---5'
EcoRV*Escherichia coli5'---GAT ATC---3'
3'---CTA TAG---5'
KpnIKlebsiella pneumonia5'---GGTAC C---3'
3'---C CATGG---5'
PstIProvidencia stuartii5'---CTGCA G---3'
3'---G ACGTC---5'
SacIStreptomyces achromogenes5'---GAGCT C---3'
3'---C TCGAG---5'
SalIStreptomyces albue5'---G TCGAC---3'
3'---CAGCT G---5'
SphIStreptomyces phaeochromogenes5'---G CATGC---3'
3'---CGTAC G---5'
XbaIXanthomonas badrii5'---T CTAGA---3'
3'---AGATC T---5'

What are the Common Use of Restriction Enzymes?

Restriction enzymes are indispensable in molecular biology and genetic engineering, particularly in molecular cloning. However, their applications are not confined solely to cloning. They are extensively applied in a broad spectrum of fields including vaccine research and development, gene sequencing, single nucleotide polymorphism (SNP) identification, ddPCR, and gene expression vectors construction. Their adaptability and precision render them essential tools across a multitude of scientific disciplines.

Gene Engineering

Type II enzymes have yielded many practical benefits, as Escherichia coli K12, its genes, and vectors have become the workhorses of molecular biology, used for cloning, library generation, DNA sequencing, enzyme and hormone detection, and overproduction. The applications of Type II enzymes continue to expand, particularly following the emergence of improved bacterial hosts and vectors for synthetic DNA, in vitro packaging of DNA into bacteriophage particles, and for protein overexpression and stability.

DNA Fingerprinting

Restriction endonucleases serve as tools for monitoring restriction fragment length polymorphisms, enabling the identification of mutations, generation of human linkage maps, and identification of disease genes (such as sickle cell trait or Huntington's disease). DNA fingerprinting can resolve cases of paternity, identify criminals and their victims, and exonerate the falsely accused. While PCR has largely replaced REases in this application, REases can be used in the system to create programs suited for such identifications. REases have also been shown to be useful in identifying pathogenic bacterial strains, with recent strains of Staphylococcus aureus exhibiting antibiotic resistance and virulence factors mediated by mobile genetic elements, such as methicillin-resistant Staphylococcus aureus (MRSA) bacteria. These strains are known to pose significant threats to humans and animals.

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