For Recombinant DNA technology we need to cut the DNA at specific sites, which are recognized and cleaved by specific enzymes, which are described as restriction enzymes.
In the year 1963, the two enzymes responsible for
restricting the growth of bacteriophage in Escherichia coli were isolated.
One of these added methyl groups to DNA, while the other cut DNA. The later was
called restriction endonuclease.
The first restriction endonuclease–Hind II,
whose functioning depended on a specific DNA nucleotide sequence was isolated
and characterised five years later. It was found that Hind II always cut
DNA molecules at a particular point by recognising a specific sequence of six
base pairs. This specific base sequence is known as the recognition sequence
for Hind II. Besides Hind II, today we know more than 900 restriction
enzymes that have been isolated from over 230 strains of bacteria each of which
recognise different recognition sequences.
Restriction enzymes belong to a larger class of
enzymes called nucleases. These are of two kinds; exonucleases and
endonucleases.
Exonucleases : Exonucleases
remove nucleotides from the ends of the DNA or RNA molecule i.e., they
catalyses hydrolysis of terminal nucleotides from the end of DNA or RNA
molecule either 5’to 3’ direction or 3’ to 5’ direction. Example: exonuclease
I, exonuclease II etc.
Endonucleases:
Endonucleases make cuts at specific positions within the DNA or RNA
molecule i.e., they can recognize specific base sequence (restriction site)
within DNA or RNA molecule and cleave internal phosphodiester bonds within a
DNA molecule. Example: EcoRI, Hind III, BamHI etc.
Each restriction
endonuclease functions by scrutinizing or inspecting’ the length of a DNA
sequence”and make the cut at the specific site called the restriction site.
Once it finds its restriction site or specific recognition sequence, it will
bind to the DNA and cut each of the two strands of the double helix at specific
points in their sugar -phosphate backbones.
The exact nature of the cut or cleavage produced by restriction
endonuclease is very important in creating a recombinant DNA. Cleavage by an
endonuclease creates DNA sequence with either a sticky end or blunt end.
Many restriction endonucleases make a simple double
stranded cut in the middle of the recognition sequence, resulting in a blunt or
flush ends.
The blunt ended fragments can be joined to any other
DNA fragment with blunt ends using linkers/adapters, making these enzymes
useful for certain types of DNA cloning experiments.
One of the
important feature of sticky-ends enzymes is that restriction endonucleases with
different recognition sequences may produce the same sticky ends. BamHI
(recognition sequence GGATCC) and BglIII (recognition sequence AGATCT) are examples, both produce GATC sticky ends
Production of these
sticky ends is the second feature of restriction enzymes that makes them
suitable for Recombination DNA technology. The principle is simple, that if two
different DNA molecules are cut with the same restriction enzymes, both will
produce fragments with the same complementary sticky ends, making it possible
for DNA chimeras to form.
Each restriction endonuclease recognises a specific
palindromic nucleotide sequences in the DNA. Pallindroms are groups of letters
that form the same words when read both forward and backward, e.g.,
“MALAYALAM”.
As
against a word-palindrome where the same word is read in both directions, the
palindrome in DNA is a sequence of base pairs that reads same on the two
strands when orientation of reading is kept the same. For example, the
following sequences reads the same on the two strands in 5' 3' direction. This is also true if read in
the 3'
5' direction. 5' —— GAATTC —— 3' ,
3' —— CTTAAG —— 5'
Which means that both strands have the same nucleotide
sequence but in anti-parallel orientation. The enzyme cuts within this
sequence.
Restriction Enzyme Nomenclature:
Restriction
endonucleases are named according to the organism in which they were
discovered, using a system of letters and numbers. The first letter of the name
comes from the genus and the second two letters come from the species of the
prokaryotic cell from which they are isolated. For example, For example,
the name EcoRI comes from Escherichia coli RY 13. E is the genus
name Escherichia, co- two letters from the species name and the letter ‘R’ is derived from the
name of strain. Roman numbers following the names indicate the order in which
the enzymes were isolated or discovered from that particular strain.
Similarly, HindIII (pronounced
“hindee-three”) was discovered in Haemophilus influenza
(strain d).
Classification of Restriction Endonucleases:
There are three major classes of restriction
endonucleases based on the types of sequences recognized, the nature of the cut
made in the DNA, and the enzyme structure:
• Type I restriction enzymes
• Type II restriction enzymes
• Type III restriction enzymes
Type
I restriction enzymes:
These enzymes have both restriction and
modification activities. Restriction depends upon the methylation status of the
target DNA.
Cleavage occurs approximately 1000 bp away
from the recognition site.
The
recognition site is asymmetrical and is composed of two specific portions in
which one portion contain 3–4 nucleotides while another portion contain 4–5
nucleotides and both the parts are separated by a non-specific spacer of about
6–8 nucleotides.
They require S-adenosylmethionine (SAM), ATP,
and magnesium ions (Mg2+) for activity.
These enzymes are composed of mainly three
subunits, a specificity subunit that determines the DNA recognition site, a
restriction subunit, and a modification subunit
Type
II restriction enzymes:
Restriction and modification are mediated by
separate enzymes so it is possible to cleave DNA in the absence of modification.
Although the two enzymes recognize the same target sequence, they can be
purified separately from each other.
Cleavage of nucleotide sequence occurs at the
restriction site.
These enzymes are used to recognize
rotationally symmetrical sequence which is often referred as palindromic
sequence.
These palindromic binding site may either be
interrupted (e.g. BstEII recognizes the sequence 5´-GGTNACC-3´, where N can be
any nucleotide) or continuous (e.g. KpnI recognizes the sequence 5´-GGTACC-3´).
They require only Mg2+ as a cofactor and ATP
is not needed for their activity.
Type II endonucleases are widely used for
mapping and reconstructing DNA in vitro because they recognize specific
sites and cleave just at these sites.
Type III restriction enzymes:
These enzymes recognize and methylate the
same DNA sequence but cleave 24–26 bp away.
They have two different subunits, in which
one subunit (M) is responsible for recognition and modification of DNA sequence
and other subunit (R) has nuclease action.
Mg+2 ions, ATP are needed for DNA cleavage
and process of cleavage is stimulated by SAM.
Cleave only one strand. Two recognition sites
in opposite orientation are necessary to break the DNA duplex.
|
Property |
Type I RE |
Type II RE |
Type III RE |
|
Abundance |
Less common than Type II |
Most common |
Rare |
|
Recognition site |
Cut both strands at a non- specific
location > 1000 bp away from recognition site |
Cut both strands at a specific, usually
palindromic recognition site (4-8 bp) |
Cleavage of one strand, only 24-26 bp
downstream of the 3´ recognition site |
|
Restriction and modification |
Single multifunctional enzyme |
Separate nuclease and methylase |
Separate enzymes sharing a common subunit |
|
Nuclease subunit structure |
Heterotrimer – Three subunits |
Homodimer – One subunit |
Heterodimer – Two subunits |
|
Cofactors |
ATP, Mg2+, SAM |
Mg2+ |
Mg2+ (SAM) |
|
DNA cleavage requirements Use in Recombinant
DNA |
Two recognition sites in any orientation Not Useful |
Single recognition site Useful |
Two recognition sites in a head-to-head orientation Not Useful |
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