The Enzymes Involved In Recombinant DNA Technology
A number of specific enzymes are utilized to achieve the objectives of rec DNA technology
The enzymology of genetic engineering includes the following types of enzymes:
(a) Restriction Endonuclease:
These enzymes serve as important tools to cut DNA molecules at specific sites, which is the basic need for rec DNA technology.
These are the enzymes that produce internal cuts (cleavage) in the strands of DNA, only within or near some specific sites called recognition sites/recognition sequences/ restriction sites
01 target sites. Such recognition sequences are specific for each restriction enzyme. Restriction endonuclease enzymes are the first necessity for rec DNA technology.
The presence of restriction enzymes was first of all reported by W. Arber in the year 1962. He found that when the DNA of phage was introduced into a host bacterium, it was fragmented into small pieces. This led him to postulate the presence of restriction enzymes. The first true restriction endonuclease was isolated in the 1970s from the bacterium E. coli by Meselson and Yuan.
Another important breakthrough was the discovery of restriction enzyme Hind-II in the 1970s by Kelly, Smith, and Nathans. They isolated it from -the bacterium Haemophilus influenza. In the year 1978, the Nobel Prize for Physiology and Medicine was given to Smith, Arber, and Nathans for the discovery of endonucleases.
Types of Restriction Endonucleases:
There are 3 main categories of restriction endonuclease enzymes:
Type-I Restriction Endonucleases
Type-II Restriction Endonucleases
Type-III Restriction Endonucleases
Type-I Restriction Endonucleases:
These are the complex type of endonucleases which cleave only one strand of DNA. These enzymes have the recognition sequences of about 15 bp length (Table 1).
They require Mg++ ions and ATP for their functioning. Such types of restriction endonucleases cleave the DNA about 1000 bp away from the 5′ end of the sequence ‘TCA’ located within the recognition site. Important examples of Type-I restriction endonuclease enzymes are EcoK, EcoB, etc.
Type-II Restriction Endonucleases:
These are the most important endonucleases for gene cloning and hence for rec DNA technology. These enzymes are most stable. They show cleavage only at specific sites and therefore they produce the DNA fragments of a defined length. These enzymes show cleavage in both the strands of DNA, immediately outs.de then- recognition sequences. They require Mg++ ions for their functioning.
Such enzymes are advantageous because they don’t require ATP for cleavage and they cause cleavage in both strands of
DNA. Only Type II Restriction Endonucleases are used tor gene cloning due to their suitability.
The recognition sequences for Type-II Restriction Endonuclease enzymes are in the form of palindromic sequences with rotational symmetry, i.e., the base sequence .n the first half of one strand of DNA is the mirror image of the second half of other strands of that DNA double helix (Fig. 2). Important examples of Type-II Restriction endonucleases include Hinfl, EcoRI, PvuII, All, Haelll, etc.
Type-III Restriction Endonucleases:
These are not used for gene cloning. They are the intermediate enzymes between Type-I and Type-II restriction endonuclease. They require Mg++ ions and ATP for cleavage and they cleave the DNA at well-defined sites in the immediate vicinity of recognition sequences, e.g. Hinf III, etc.
Nature of cleavage by Restriction Endonucleases;
The nature of cleavage produced by a restriction endonuclease is of considerable importance. They cut the DNA molecule in two ways:
- Many restriction endonucleases cleave both strands of DNA simply at the same point within the recognition sequence. As a result of this type of cleavage, the DNA fragments with blunt ends are generated. PvuII, Hall, All are examples of restriction endonucleases producing blunt ends. Blunt ends may also be referred to as flush ends.
- In the other style of cleavage by the restriction endonucleases, the two strands of DNA are cut at two different points. Such cuts are termed as staggered cuts and this results in the generation of protruding ends i.e., one strand of the double helix extends a few bases beyond the other strand. Such ends are, called cohesive or sticky ends.
Such ends have the property to pair readily with each other when pairing conditions are provided. Another feature of the restriction endonucleases producing such sticky ends is that two or more of such enzymes with different recognition sequences may generate the same sticky ends.
Exonuclease is an enzyme that removes nucleotides from the ends of a nucleic acid molecule. An exonuclease removes nucleotide from the 5′ or 3′ end of a DNA molecule. An exonuclease never produces internal cuts in DNA.
In rec DNA technology, various types of exonucleases are employed like Exonuclease Bal31, E. coli exonuclease III, Lambda exonuclease, etc.
Exonuclease Bal31 is employed for making the DNA fragment with blunt ends shorter from both its ends.
E Coli Exonuclease III is utilized for 3’end modifications because it has the capability to remove nucleotides from the 3′-OH end of DNA.
Lambda exonuclease is used to modify 5′ ends of DNA as it removes the nucleotides from the 5′ terminus of a linear DNA molecule.
The function of these enzymes is to join two fragments of DNA by synthesizing the phosphodiester bond. They function to repair the single-stranded nicks in DNA double helix and in rec DNA technology they are employed for sealing the nicks between adjacent nucleotides. This enzyme is also termed as a molecular glue.
These are the enzymes that synthesize a new complementary DNA strand of an existing DNA or RNA template. A few important types of DNA polymerases are used routinely in genetic engineering. One such enzyme is DNA polymerase! which, prepared from E Coli. The Klenow fragment of DNA polymerase-I .s employed to make the protruding ends double-stranded by extension of the shorter strand.
Another type of DNA polymerase used in genetic engineering is Taq DNA polymerase which is used in PCR (Polymerase Chain Reaction).
Reverse transcriptase is also an important type of DNA polymerase enzyme for genetic engineering. It uses RNA as a template for synthesizing a new DNA strand called cDNA an e complementary DNA). Its main use is in the formation of cDNA libraries. Apart from all these above-mentioned enzymes, a few other enzymes also mark their importance in genetic engineering.
A brief description of these is given below:
- Terminal deoxynucleotidyl transferase enzyme:
- It adds single-stranded sequences to the 3′-terminus of the DNA molecule. One or more deoxyribonucleotides (dATP, dGTP, dl IP, dCTP) are added onto the 3′-end of the blunt-ended fragments.
- Alkaline Phosphatase Enzyme:
It functions to remove the phosphate group from the 5′-end of a DNA molecule.
- Polynucleotide Kinase Enzyme:
It has an effect reverse to that of Alkaline Phosphatase, i.e. it functions to add a phosphate group to the 5′-terminus of a DNA molecule