3. • A Restriction Enzyme or Restriction Endonuclease is a bacterial enzyme that cuts dsDNA into
fragments after recognition specific nucleotide sequence known as recognition or restriction site.
• Restriction enzymes are produced by bacteria as a defense mechanism against infection by
viruses. They restrict or prevent viral infection by degrading the DNA of invading viruses.
• The term restriction comes from the fact that these enzymes restrict the entry of foreign DNA in
the bacteria.
• Restriction enzymes, are believed to be evolved by bacteria to resist viral attack.
Restriction Enzyme
Introduction
History
• The existence of restriction enzymes was first postulated by W. Arber.
• He noticed that when the DNA of a bacteriophage entered a host bacterium it was cut up into
smaller pieces and, for this, he theorized the presence of restriction enzyme.
• In 1970, Hamilton Smith and his co-workers first isolated a restriction enzyme from the
bacterium Haemophi/us influenzae strain Rd.
• The enzyme, called Hindll, recognizes a six base-pair dsDNA sequence. After discovery of
Hindll restriction enzyme, EcoRI, was isolated and characterized from Escherichia coli strain
4. Nomenclature
The name of any restriction endonuclease consists of three parts:
1. An abbreviation of the genus and species of the organism to three letters, e.g. Eco for
Escherichia coli identified by the first letter of the genus and the first two letters of the species.
2. A letter, number or combination of the two to indicate the strain of the relevant species.
3. A Roman numeral to indicate the order in which different restriction modification systems were
found in the same organism or strain.
For example, the name of the EcoRI restriction enzyme was derived as:
E Escherichia (genus)
co coli (species)
R RY13 (strain)
I First identified (order of identification in the bacterium)
5. Restriction Site
• A restriction enzyme recognizes and binds to DNA at a specific nucleotide sequence called a
restriction site
• The enzyme then cuts both strands of the DNA within that sequence by cleaving the
phosphodiester backbone of DNA.
• Most recognition sequences exhibit a form of symmetry described as a palindrome: the
nucleotide sequence reads the same on both strands of the DNA when read in the 5’ to 3’
direction.
WAS IT A RAT I SAW
राधा की बूनी में नीबू की धारा
• The position at which the restriction enzymes cuts is usually shown by the symbol ‘/’.
• Enzymes such as EcoRI and HindIII make offset cuts in the DNA strands, thus producing
fragments with single-stranded overhanging ends called cohesive ends, while others such as
AluI and BalI cut both strands at the same nucleotide pair, producing DNA fragments with
double stranded ends called blunt-end fragments.
6.
7. Star Activity
Under non-standard reaction conditions
(such as low ionic strength, high pH), some
restriction enzymes are capable of cleaving
sequences which are similar but not
identical to their defined recognition
sequence. This altered specificity has been
termed star activity.
• Isoschizomers are pairs of R.E. That recognize the same recognition sequence and cut in same
location. For example, Sphl and Bbul (CGTAC/G) are isoschizomers of each other.
• Neoschizomers are enzymes that recognize the same sequence, but cut it differently. For
example, Smal (GGG/CCC) and XmaI (G/GGCCC) are neoschizomers of each other.
• Isocaudomers are enzymes that recognize slightly different sequence, but produce the same
ends. For example, both Sau3A (N/GATC) and BamHl (G/GATCC), gives a 5’-GATC-3’ sticky
end and have different recognition sequence.
8. Frequency of recognition sites
If it is assumed that the nucleotides are ordered in a random fashion and that the
four different nucleotides are present in equal proportion then the recognition site
of 6 bp will occur at a frequency of (¼)6 i.e. 1/4096 bases.
So, EcoRl, which recognize the sequence of 6bp(GAATTC).
If the GC content of the genome DNA is supposed to be 40%, then AT will be 60%.
Hence, G and C will be 20% each while A and T will be 30% each. For restriction
site AAGCTT, the probability of occurrence will be
3/10 × 3/10 × 2/10 × 2/10 × 3/10 × 3/10 = 324/1000000 =
~1/3086
9. References
1. Life sciences Fundamentals and Practice ll, Edition 6th , Pathfinder
publication.
2. Concept of genetics Tenth edition, klug, Cummins, et al.
Thank You
