2. • Restriction Enzymes:
• Steward Linn and Werner Arber (1963) isolated two enzymes which restricted the
growth of bacteriophage in bacterium E. coli. One of these enzymes added methyl
groups to DNA and second one cut DNA. The second enzyme was named
as “restriction endonuclease.”
• H.O. Smith, K.W. Wilcox and T.J. Kelley (1970) isolated restriction
endonuclease whose working depended on a particular nucleotide
sequence. They isolated this enzyme from bacteria Haemophilus
influenzae and called is as Hind II. It was observed that Hind II always cut
DNA molecules at specific place by identifying a particular sequence of six
base pairs.
• Restriction enzymes belong to a larger class of enzymes called nucleases.
3. • They are of two types:
• (i) Exonucleases:
• They remove nucleotides from the ends of DNA.
• (ii) Endonucleases:
• They make cuts at specific positions within DNA.
• Thus, a restriction enzyme (or restriction endonuclease) recognizes a specific base pair sequence in
DNA called a restriction site and cleaves the DNA (hydrolyzes the phosphodiester back bones)
within the sequence. Restriction enzymes are widely found in prokaryotes and provide protection to
host cell by destroying foreign DNA that makes entry into it.
• Here they act as a part of defence mechanism called Restriction Modification System.
• It bears two components:
• (a) First component is a restriction enzyme that selectively identifies a specific DNA sequence and
degrades any DNA bearing that sequence.
4.
5. • (b) In second component is a modification enzyme. It adds a methyl group to one or two
bases within the sequence identified by restriction enzyme. If a base in DNA is modified
due to addition of methyl group, restriction enzyme cannot identify and cleave that DNA.
By this method bacteria are able to protect their chromosomal DNA from cleavage by
restriction enzymes.
• Thus, bacteria bear sets of restriction endonucleases and corresponding methylases.
• Endonucleases are enzymes that produce internal cuts called cleavage in DNA molecules.
Endonucleases cleave DNA molecules at random sites. A class of endonucleases cleaves
DNA only within or near those sites with specific base sequences called restriction
endonucleases. Sites recognised by them are called recognition sites or recognition
sequences. These sites differ for different restriction enzymes.
• Restriction endonucleases serves as the tools for cutting DNA molecules at predetermined
sites, which is the basic requirement for gene cloning or recombinant DNA technology.
6.
7. • Types of Restriction Endonucleases:
• Three main types of restriction
endonucleases i.e., Type I, Type II and
Type III are known with slightly different
mode of action. Type II restriction
enzymes are used in rDNA technology
because they can be used in vitro to
identify and cleave within specific DNA
sequences usually having 4-8 nucleotides.
• More than 350 different type II
endonucleases with 100 different
recognition sequences are known. They
need Mg2+ ions for cleavage. The first type
II enzyme isolated was Hind II in 1970.
8. The recognition sequences for Type II restriction enzymes form pallindromes with
rotational symmetry. In a pallindrome, base sequence of second half in DNA strand
represents the mirror image of the base sequence of first half. Due to this in DNA double
helix, complementary strand also represents the same mirror image.
Pallindromes are groups of letters that form the same words when read both forward and
backward e.g., ‘MALAYALAM’. As against a pallindrome when same word is read in both
the directions, pallindrome in DNA is a sequence of base pairs that reads same on the two
strands when orientation of reading is kept the same.
9. • However in pallindromes with rotational symmetry, second half of complementary strand
in DNA double helix is the mirror image of base sequence in the first half of another
strand. In such cases, base sequences in both the strands of DNA helix represents the
same when read from same and i.e., either 5′ or 3′ of both strands in DNA duplex, e.g.,
• Eco RI cleaves DNA molecule of two plasmids due to similar recognition sites in their
DNA. The circular form of DNA becomes linear in both the cases. Such linear DNAs can
stick together to form single recombinant DNA molecule.
10. Nomenclature:
Nomenclature of restriction enzymes is usually done by following technique:
(i) The first letter of the genus is taken in which said enzyme was discovered. This letter is
written in capital.
(ii) Then, first two letters of species of that organism are written.
(iii) All the above three letters should be written in italics.
Examples:
Eco from Escherichia coli, Hin from Haemophilus inflenzae and Hpa from Haemophilus
parainfluenzae.
(iv) This followed by strain or type identification e.g., Eco K.
(v) When the enzyme is encoded by plasmid, the name of plasmid is written e.g. Eco RI i.e.,
Eco RI comes from Escherichia coli RY13. Here ‘R’ is derived from the name of strain.
Roman numbers following the names indicate the order in which enzymes were isolated
from the strain of bacteria.
11. • (vi) If an organism forms many enzymes, they are identified by sequential Roman
numerals.
• Example:
• Enzymes formed by H. influenzae strain RD have been named as Hin II, Hin III etc.
• Discovery of Enzyme Eco RI led to award of Nobel Prizes to W. Arber, H. Smith and
D. Nathans in 1978.
• Types of Cleavage Produced By Restriction Enzymes:
• Many restriction enzymes like Smal isolated from Serratia marcescens cleave both the
strands of DNA at exactly same nucleotide position almost in centre of recognition
site resulting in blunt or flush end.
• Smal recognizes the 6 nucleotide palindromic sequence and cleave at both the ends.
12. • Still some other restriction enzymes cleave the recognition
sequence asymmetrically. Thus due to cleavage, they produce
short, single stranded hanging structures. Such ends are called
sticky or cohesive ends because base pairing between them can
stick the DNA molecule again. A 6 nucleotides palindromic
nucleotide sequence recognised by Eco RI cleave both strands at
different points.
