Restriction Endonucleases | Molecular Scissors | Biology Neet

 Restriction Endonucleases | Molecular Scissors

• Restriction endonuclease was isolated for the first time by W. Arber in 1962 in bacteria.
• They are called " molecular scissors or biological scissors ”.
• In 1978 Arber, Smith and Nathan were awarded the Nobel Prize for the discovery of restriction endonuclease.
• They recognize the base sequence at palindrome sites in DNA duplex and cut its strands.
• For example, restriction endonuclease EcoR I found in the colon bacteria E.coli, recognizes the base sequence GAATTC in DNA duplex and cuts its strands between G and A as shown below

• Only restriction enzymes type II are used in gene manipulation for two reasons.
• No ATP is needed for the cleaving action.
• It makes cleavage or cut in both the strands of DNA molecule.
• Types of Restriction Endonucleases
• Three main types of restriction endonu cleases are type I , type II and type III.
• Type I Restriction Endonucleases
• These enzymes consist of 3 different subunits.
• They require ATP, Mg2 + and S - adenosyl methionine for restriction.
• Type I restriction endo nucleases recognize specific sites within the DNA but do not cut these sites.
• Thus they are not used in recombinant DNA technology.
• Type II Restriction Endonucleases.
• These enzymes are simple and require Mg2 + ions for restriction.
• Out of the three types , only type II restriction enzymes are used in recombinant DNA technology.
• Type III Restriction Endonucleases
• These enzymes consist of two different subunits.
• They require ATP, Mg2 + and S - adenosyl methionine for restriction.
• They recognize specific sites within DNA but do not cut these sites, therefore, these restriction endonucleases are not used in recombinant DNA technology.
• They have intermediate properties between type I and type II.

Nomenclature of Restriction Enzymes

• Type II restriction enzymes are named Kor the bacterium from which they have been isolated.
• The first letter used for the enzyme is the first letter of the bacterium's genus name in italics.
• Then comes the first two letters of its species (also in italics).
• The fourth letter of the name of enzyme is first letter of the strain . It is written in capital.
• The end of the name indicates the order in which the enzyme was isolated
• It is written in Roman number.
• For example, the enzyme Eco RI was isolated from the bacterium Escherichia coli RY13.
• Enzyme Eco Rl is named as follows.
• The capital letter E comes from the genus Escherichia.
• The letters co are from the species coli.
• The letter R is from RY13 (strain).
• The Roman number 1 indicates that it was the first enzyme isolated from the bacterium E.coli RY13.
• The discovery of restriction endonuclease enzymes led to Nobel Prize for W. Arber , H. Smith and D. Nathan in 1978.
• Some examples of type II enzymes are given in the table showing names of restriction enzymes ( endonucleases ) , source ( organism from where they have been isolated ) , their recognition sequence and site of cleavage.

Functioning of Restriction Endonuclease

• The foundations of recombinant DNA (rDNA) were laid by the discovery of restriction enzymes.
• These enzymes are present in many bacteria where they function as a part of their defence mechanism called the Restric tion Modification System.
• Molecular basis of this system was explained first by Wemer Arber in 1965.

• The Restriction - Modification system consists of two components
• A restriction enzyme which identifies the introduced foreign DNA and cuts into pieces called restriction endonucleases.
• The term ' restriction ' refers to the function of these enzymes in restricting the propagation of foreign DNA of bacteriophages (viruses that attack bacteria ) in the host bacterium.
• The second component is a modification enzyme that adds a methyl group to one or two bases usually  within the sequence reorganized by the restriction enzyme.
• Once a base in a DNA sequence is modified by addition of a methyl group , the restriction enzymes fail to recognize and could not cut that DNA.
• This is how a bacterium modifies and therefore, protects its own chromosomal DNA from cleavage by these restriction enzymes.
• The first restriction endonuclease was Hind II (hin-dee-two).
• Its functioning depended on a specific DNA nucleotide sequence.
• It was isolated from Haemophilus influenzae Rd.
• It was found that Hin d 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 recog nition sequence for Hin d II.
• It produces blunt ends.Besides Hin d II, today we know more than 900 restriction enzymes that have been isolated from over 230 strains of bacteria each of which recognises different recognition sequences

Palindromic Nucleotide Sequence

• The restriction endonuclease inspects the length of a DNA sequence.
• Once it recognises specific sequence, it binds to the DNA and cuts each of the two strands of the double helix at specific points in their sugar phosphate back bones.
• Special sequence in the DNA recognised by restriction endonuclease is called palindromic nucleotide sequence.
• Restriction endonuclease recognizes palindromic sequences in DNA and cuts them.
• The palindromes are groups of letters that form the same words when read in both directions forward and backward.

• The palindromes in DNA are base pair sequences that are the same when read forward (left to right) or backward ( right to left ) from a central axis of symmetry.
• For example, the following sequences read the same on the two strands in 5 ' 3 ' direction.
• This is also true when we read in the 3'5 ' direction.
• Restriction enzymes cut the strand of DNA a little away from the centre of the palin drome sites but between the same two bases of the opposite strands.
• This leaves single stranded unpaired bases at cut ends.
• These ends with unpaired bases are called sticky ends or cohesive ends
• The latter are named so because they form hydrogen bonds with their complementary cut counterparts.
• The sticky ends facilitate the action of the enzyme DNA ligase.

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