In this article we will discuss about:- 1. Meaning of Restriction Enzymes 2. Naming of Restriction Enzymes 3. Mechanism of Action.
Meaning of Restriction Enzymes:
These are also known as molecular scissors, used for cutting of DNA. The cutting of DNA at specific locations became possible with the discovery of molecular scissors, i.e., restriction enzymes. In the year 1963, the two enzymes responsible for restricting the growth of bacteriophage in E. coli were isolated. One of these added methyl groups to DNA, while the other cuts the DNA. Later was termed as restriction endonucleases.
The first restriction endonuclease was isolated by Smith Wilcox and Kelley in 1968 was Hind II. It was found that it always cuts DNA molecules at a particular point by recognising a specific sequence of six base pairs known as recognition sequence for Hind II. Today, more than 900 restriction enzymes have been isolated from over 230 bacterial strains each of which recognise different recognition sequences.
Naming of Restriction Enzymes:
The convention for naming these enzymes proceeds in a way that the first letter of the name comes from the genes and the second two letters come from the species of prokaryotic cell, from which they were isolated, e.g., Eco RI comes from E. coli RY13. The letter ‘R’ is derived from the name of strain. Roman numbers following the names, indicate the order in which the enzymes were isolated from that strain of bacteria.
Restriction enzymes belong to a larger class of enzymes called nucleases, which are of two types:
(a) Exonucleases remove nucleotides from the ends of the DNA either (5′ or 3′) in one strand of duplex.
(b) Endonucleases make cuts at specific positions within DNA. Each restriction endonucleases function by ‘inspecting’ the length of a DNA sequence.
Once it finds its 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.
Differences between Exonucleases and Endonucleases
Exonucleases:
1. These nucleases cleave base pairs of DNA at their terminal ends
2. They act on single – strand of DNA or gaps in double –stranded DNA. They do not cut RNA
Endonucleases:
1. They cleave DNA at any Pont except the terminal ends
2. They cleave one strand (figure below) or both strands (figure below) of double – stranded DNA. They may cut RNA
Each restriction endonuclease recognizes a specific palindromic nucleotide sequences in the DNA.
Palindrome in the DNA is a groups of letters that forms the same words when read both forward and backward.
For example, the following sequences read the same on the two strands in 5′ → 3′ direction as well as 3′ → 5′ direction.
Mechanism of Action of Restriction Enzymes:
Restriction enzymes cut the strand of DNA a little away from the center of the palindrome sites, but between the same two bases on the opposite strands. This leaves single-stranded portion at the ends. There are overhanging stretches called sticky ends on each strand as given in above figure.
Restriction endonucleases are also used in genetic engineering to form recombinant molecules of DNA, which are composed of DNA from different sources or genomes. The resultant DNA fragments have the same sticky ends, which are complementary to each other, therefore can be joined together (end-to-end) using DNA ligases, when cut by the same restriction enzyme.
In order to create a recombinant vector molecule, it is necessary that the vector and the source DNA should cut with the same restriction enzyme.