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Restriction enzymes are one of the few tools necessary for Recombinant DNA Technology or Genetic engineering. Other tools required are Vectors, Host organism, Polymerase enzymes, Ligases.
Discovery of Restriction enzymes
As we know that Bacteriophage is a virus capable of infecting bacteria (Escherichia coli), but in year 1963, few strains of bacteria were found to be immune to bacteriophage infection. These bacterial strains had restricted the growth of bacteriophage in them. After observation it was found that restriction occurs because the bacteria produces an enzyme that degrades the phage DNA before it can replicate inside bacteria and synthesize new phages. Bacteria protect its own DNA from attack by these degradative enzymes by addition of methyl groups to its DNA. These degradative enzymes are called as Restriction enzymes or Restriction endonucleases.
HindII is the first restriction endonuclease discovered.
Nomenclature of Restriction endonucleases
First letter of the name of these enzymes comes from Genus of the host organism (prokaryotic cell) from which the enzyme is isolated. Second two letters comes from species of that prokaryotic cell. This abbreviation is always written in italics.
Next letter in non-italics is the particular strain of that host organism or prokaryotic cell from which it is isolated. Roman number indicates the order of discovery of enzyme from that strain of prokaryotic cell.
- HindII : isolated from Haemophilus influenzae, strain d, 2nd enzyme
- HindIII : isolated from Haemophilus influenzae, strain d, 3rd enzyme
- EcoRI : isolated from Escherichia coli, strain RY 13, 1st enzyme
Working of Restriction endonucleases
Restriction enzymes belong to a larger class of enzymes called Nucleases. Nucleases are enzymes that cut, shorten, or degrade nucleic acid molecules. Nucleases degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand. These are of two kinds: Exonucleases and Endonucleases.
- Exonucleases: these remove nucleotides one at a time from the ends of the DNA molecule
- Endonucleases: these break / cut internal phosphodiester bonds at specific positions within a DNA molecule
So each restriction endonuclease cuts a DNA molecule at a specific position. This specific position is determined by specific sequence of base pairs present in DNA. This specific base sequence is known as Recognition sequence.
Recognition sequence is a four to eight nucleotide long sequence and is always palindromic. Palindromic sequence is that sequence which can be read same in both forward and backward direction. e.g. in general terms word “MALAYALAM” same in both direction, “NITIN” also same.
In case of DNA , palindromic sequences are those sequences of nucleotides which are read same on both strands of DNA when read in same orientation either 5′ to 3′ or 3′ to 5′. e.g. nucleotide sequence on one strand 5′ GAATTC 3′ and on another strand nucleotide sequence 3′ CTTAAG 5′.
So each restriction endonuclease inspects the length of a DNA sequence for palindromic recognition sequence before cutting the DNA .Once it finds its specific recognition sequence, it binds to the DNA and cut each of the two strands of the double helix at that specific point.
Each restriction endonuclease has its specific recognition sequence and it cuts DNA at that palindromic recognition sequence only, nowhere else. Like EcoRI has recognition sequence 5’GAATTC3’, HindIII has recognition sequence 5’AAGCTT3’.
Restriction enzymes cut the strands of DNA in two ways:
1. At the exact center of recognition sequence which produces Blunt ends or Flush ends.
2.Little away from the centre of the recognition sequence, but between the same two bases on opposite strands. This leaves short single stranded portions / overhangs on each strand. These overhanging portions are called Sticky ends or Cohesive ends on each strand.These are named so because they form hydrogen bonds with their complementary cut counterparts.
Ligase enzyme helps in joining two same sticky ends produced by the same restriction endonuclease as specific restriction endonuclease produces same sticky ends in every situation.
Restriction endonuclease are used to create same sticky ends in vector and foreign DNA so that they can be joined by ligase enzyme to create recombinant DNA molecule.
DNA fragments produced by cuts of restriction endonuclease can be separated by Agarose Gel Electrophoresis.