The nuclease enzyme that cuts the DNA at a unique sequence, is called restriction endonuclease.
They cut the DNA in a non-terminal region. Restriction endonucleases are used to generate rejoinable DNA fragments.
They are also known as molecular knives, molecular scissors, restriction enzymes or molecular scalpels.
The sequence recognized by the restriction enzyme to cut the DNA is called restriction site, restriction endonuclease site or recognition site. The recognition site consists of 4-8 base pairs.
The enzyme breaks two phosphodiester bonds, one in either strand of the duplex DNA to cut the DNA. The 3' cut end has a free-OH group and the 5' cut end has a phosphate end.
Some restriction enzymes recognize palindromic sequences to cut DNAs, but some others recognize non-palindromic sequences.
The genome of an organism has several restriction sites for one restriction enzyme. The distance between two adjacent restriction sites varies greatly. So a restriction enzyme produces several DNA fragments of different lengths while cutting the DNA.
The restriction endonucleases are grouped into three types-
• Type I endonucleases
• Type II endonucleases
• Type III endonucleases
The Type I and III restriction enzymes recognize specific sequences in the duplex DNA but cut the DNA far away from the recognition sites. So they are not useful for genetic engineering.
The type II restriction endonucleases recognize specific sites and cut the DNA at the recognized sites. So they are of much use in genetic engineering e.g. Eco RI, Hind III, etc.
A type II restriction endonuclease recognizes a specific sequence in the duplex DNA and cuts the DNA at the recognized sequence. So the cutting is sequence specific. The enzyme consists of two identical sub- units and its molecular weight ranges from 20000 to 100000 daltons. It requires Mg2+ as co- factor for the enzyme activity. Eco RI, MboI, etc. are examples for type II restriction enzymes.
At present, about 350 type II restriction endonucleases are isolated from various bacterial strains. They are named using the first letter of the genus name, the first two letters of the species name and the first letter of the strain from which the enzyme was isolated. If there are more than one restriction enzyme in a strain, they are designated in Roman numeral. For example, EcoRI is isolated from Escherichia coli RY 13. The final number I indicates that it is the first enzyme isolated from the strain.
The type II res • triction enzymes mostly recognize palindromic sequences to cut the DNA. The palindromic sequence consists of 4-6 basepairs and is bilaterally symmetrical. The base sequence in one strand is the same in the other strand while reading in reverse direction. An axis or line cuts the palindromic sequence into two identical halves, and is called axis of symmetry. Some restriction enzymes cut at the restriction site along the axis of symmetry while others cut it at either side of the axis of symmetry.
Some restriction enzymes cut DNAs along the axis of symmetry of the restriction sites. They break two phosphodiester bonds, one in either strand of the restriction site, at the axis of symmetry. Hence two blunt -ends are formed.
Several restriction enzymes cut one strand at left side of the axis of symmetry and the other at the right side of the axis. Then they break hydrogen bonds between base pairs lying between the two cut-sites. As a result, DNA fragments with single stranded extensions are formed. The single stranded extensions are called cohesive ends or sticky ends. The sticky ends of the DNA fragments produced by a restriction enzyme are complementary to each other. The 5'- position of cut end has a phosphate group and the 3'- position has an OH-group. E.g. EcoRI, Bam HI, etc.
Restriction enzymes are used to cut a source DNA into small fragments for the isolation of a desired gene to be cloned
• They are used to cutout unwanted sequences from natural vector DNAs to construct active vectors.
• They are used to cut large DNA into small fragments for nucleotide sequencing.
• They are used to construct restriction map of DNAs.
• They are used to cut DNAs to determine variant sequences among the DNAs of closely related individuals by restriction fragment polymorphism (RFLP).
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