Restriction Endonucleases

Discovery

• In his 1969 paper, Werner Arber proposed that host-controlled restriction in bacteriophages occurs as result of enzymatic cleavage of infecting DNA molecules by what he termed as endonuclease while refering to endonuclease R (later to be renamed as EcoB)
• Arber, along with Struat Linn received the Nobel Prize in Physiology and Medicine in 1978 for the discovery of restriction "enzymes" or "endonucleases"
• They also proposed that DNA modification is the result of DNA methylation by DNA "methylases"
• Shortly after the discovery, another restriction enzyme was found in E. coli (EcoK)

Nomenclature

• There is a standard set of rules followed for nomenclature of the enzymes:
• The first three letters of the name are an abbreviation of the name of the species of bacteria from which it has been isolated
• The fourth letter refers to the strain of the bacterium
• Roman numerals are used to denote the enzymes when more than one has been isolated from a single source
• Example: EcoRI, where Eco stands for E. coli

Restriction Endonucleases have been found to be of 4 types:

Type I

• Complex
• Found in EcoB, EcoK, phage-encoded enzyme complex P1
• Endonuclease functions simultaneously as an endonuclease + methyltransferase
• Requires Mg2+, ATP and SAM as co-factors
• Restriction and modification activities are present on different subunits
• RE → stretches of DNA 15 bp in length, methylated or cleaved at positions approximately 1000 bp away from the 5' TCA sequence of the recognition site
• DNA sequences methylated at both As on either strand are resistant
• Heteroduplexes are ideal substrates where unmodified DNA is cleaved
• Only recognition sites and not cleavage sites display specificity and therefore of little importance in Genetic Engineering

Type II

• DNA binding sites coincide with cleavage sites and are specific
• Stable, requiring only Mg2+ as a co-factor
• RE → tetra, penta, or hexanucleotides having axis of rotational symmetry (i.e. palindromic)
• Enzymatic activities of these endonucleases can be detected by:
• measuring loss of biological activity
• observing changes in viscosity or sedimentation behavior of DNA preparations
• The number of Type II sites in a DNA molecule depends on:
• size of DNA
• base composition
• GC content

Type III

• Cleave DNA at specific sequences in the vicinity of recognition sequence (approximately 25-27 bp downstream)
• Require ATP as well as Mg2+ as a co-factor
• Lack ATPase activity and requirements of SAM
• Produce homogenous populations of DNA fragments