What are Restriction Enzymes?
The restriction enzyme is a protein produced by bacteria that cleaves the DNA at specific sites. This site is known as the restriction site.
The restriction enzymes protect the live bacteria from bacteriophages. They recognize and cleave at the restriction sites of the bacteriophage and destroy its DNA.
Restriction enzymes are important tools for genetic engineering. They can be isolated from the bacteria and used in the laboratories.
The restriction enzymes recognize short and specific nucleotide sequences in the DNA known as the recognition sequences When the restriction enzyme recognizes a DNA sequence, it hydrolyzes the bond between adjacent nucleotide and cuts through the DNA molecule.
The bacteria prevents its own DNA sequences from degradation by the addition of the methyl group at the adenine or cytosine bases within the recognition sequence with the help of enzyme methylases.
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Types of Restriction Enzymes
These restriction enzymes cut the DNA far from the recognition sequences. However, they do not produce discrete restriction fragments, hence, are of not much practical value.
These are complex, multisubunit restriction and modification enzymes. They were initially thought to be rare, but through genomic analysis, they are found to be common and are of considerable biochemical interest.
These enzymes cut at specific positions closer to or within the restriction sites. Discrete restriction fragments and gel banding patterns are observed. They are exclusively used for DNA analysis and gene cloning in the laboratories. These are a family of unrelated proteins. They are named after the bacterial species from which they are isolated. For eg., EcoRI is isolated from bacterial species E.coli. The restriction enzymes generate two different types of cuts. Blunt ends are produced when they cut the DNA at the centre of the recognition sequence, and sticky ends produce an overhang.
These are multifunctional proteins with two subunits- Res and Mod. It is a modification methyltransferase. The DNA sequence specific for the system is recognized by the Mod subunit.
Applications of Restriction Enzymes
They are used in RFLP techniques to cut the DNA into smaller fragments to study the fragment length differences among the individuals.
In Gene Cloning
During cloning, a gene is inserted into a plasmid. Restriction enzymes cut the plasmid producing single-stranded overhangs. The two DNA molecules are ligated with the help of DNA ligase to form a single DNA molecule.
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Q.1. How is a restriction site recognized by the restriction enzymes?
A.1. Restriction enzymes are isolated from the prokaryotes. They recognize the specific DNA sequences and bind to it. These are known as restriction sites. The enzyme makes a double-stranded cut in the molecule on encountering its target sequence.
Q.2. What are exonucleases?
A.2. The enzymes that remove nucleotides from the end of the DNA one at a time are known as exonucleases.
Q.3. How does a restriction enzyme protect its own DNA from cleavage?
A.3. The restriction enzyme methylates its DNA so that it is not recognized by the restriction endonuclease. Thus they prevent the restriction enzyme from cutting its own DNA.
Q.4. How does the modified DNA remain protected after replication?
A.4. One of the DNA strands remains methylated even after replication due to the semi-conservative mode of DNA replication. One methylated strand can protect the DNA from cleavage by restriction enzymes.
Q.5. How many types of restriction enzymes are there?
A.5. There are three types of restriction enzymes- Type I, II, III.