ENZYMES USED FOR RECOMBINANT DNA TECHNOLOGY

ENZYMES USED FOR RECOMBINANT DNA TECHNOLOGY

7.   ENZYMES USED FOR RECOMBINANT DNA TECHNOLOGY 

Two enzymes main role play in RDT
1.    DNA ligase
2.    Restriction endonuclease    
1.    DNA LIGASE 
DNA ligases are the enzymes which facilitate the ligation of DNA strands by the formation of the phosphodiester bond. In recombinant DNA technology, it is used to ligate the desired DNA segment at the target site of the vector.
2.    Restriction Endonuclease
These are enzymes which have the ability to catalyze the cleavage of DNA at specific sites to produce fragments of DNA. The site recognized and cleaved by a restriction endonuclease is called its restriction site.
The major class of restriction endonuclease
The restriction endonucleases are grouped on the basis of sequence it recognises, the nature of cut made on DNA and the structure of the enzyme. Type I and Type III cleaves the DNA at sites other than the restriction site and produce random cleavage, therefore, are not much use for gene cloning. Type II is the most used restriction endonuclease for gene cloning as they cleave the DNA at specific sites only. 
Type II restriction endonuclease: Type II restriction endonuclease is typically a homodimer composed of two identical polypeptide subunits. Most of Type II restriction endonuclease has the common structural core and consists of four conserved b-strands and one a-helix. It also requires cofactor Mg+2 ion. The enzyme recognizes short sequences of DNA of around 5-8 bp. This sequence is read in 5' to 3' direction on one strand and the same sequence is studied in 5' and 3' direction in the complementary strand. These sequences which are read the same in both directions are known as a palindromic sequence.
This enzyme cleaves both the strand at the same time which is in close proximity to recognizing the site. The enzyme cleaves the DNA by breaking the covalent phosphodiester bond between phosphate group of one nucleotide and sugar of adjacent nucleotide and produce free 5' phosphate and 3' OH. There is no energy utilisation enzyme in this process. EcoRI generates a staggered cut which produces single-stranded DNA at one end. This single-stranded DNA can form a hydrogen bond with a complementary sequence produced by some enzyme in other DNA. These single stood complementary tails are known as sticky or cohesive ends.
On the other hand type II restriction, endonuclease-like smaf cleaves at the same position and generate blunt ends with no unpaired nucleotides. Restriction endonucleases are very specific in recognizing the sequence and in case of a mutation in even a single nucleotide can lead to loss of the enzymatic activity of the enzyme.

The restriction endonuclease first binds non-specifically at DNA without interacting with bases but interacts only with sugar-phosphate backbone. The catalytic centre is at safe distance from DNA phosphate backbone and binding is very loose. This enzymes then walk over DNA to search for a specific site. For example, BamHI moves linearly over the DNA. this is known as sliding. Sliding is basically helical movement along the DNA due to tracking along the groove of DNA over the short distance. When the target site is recognised there is a conformational change in the enzyme and DNA. This activates the catalytic centre for catalysis. 

Natural restriction system in bacteria
A study was performed to observe the cycle of bacteriophage in different strains of bacteria. The study was done on l phage and it was first allowed to grow on C strain of Escherichia coli. But this same phage is restricted in k-12 strain (the standard strain of E.coli for molecular biology studies). However, some rare l phage managed to grow on K-12 strain. These l phage is known as k-modified. These rare phages can grow normally on both C and K strain of K-12. But after growth on C, the K modified phage is again restricted in K-12. This shows that own DNA of K-12 remain preserved while other DNA from distantly related strain is eliminated.
This experiment gave a clue that there must be a nuclease that can distinguish between resident and foreign DNA. After some years, this nuclease was extracted from the K-12 strain of E.coli. This nuclease cleaved the modified phage DNA into about five fragments but did not affect the modified phase. This enzyme was named restriction nuclease because they prevent viral infection by clearing the viral DNA.
Modification System
Further research leads to an inference that addition of methyl group at the sites which are sensitive to the attack of restriction enzyme can protect the DNA from cleavage. Generally, in E.coli, the addition of the methyl group at adenine (7-methyladenine) is more common than cytosine methylation (6-methyl cytosine).
The restriction enzymes are not able to recognize these modified sites and hence DNA is not degraded. This modified pattern is also maintained in replication. When the self DNA replicates, the older strand remains methylated while the new strand is not methylated. This hemimethylated state is quickly recognized and the new strand is methylated by specific methylated enzymes. On the contrary, foreign DNA is in unmethylated from and therefore is degraded.


Recombinant DNA Applications
1.    In Vitro Mutagenesis: It is possible (and relatively easy) to make specific mutations in a gene using a variety of methods which are collectively called site-directed mutagenesis.
2.    Gene synthesis: It is possible to synthesize small segments of DNA with a particular nucleotide sequence. These segments are called oligonucleotides.
3.    Expressing eukaryotic genes in bacteria: eg. Insulin–1st recombinant product to be licensed for therapeutic use in 1982. Other examples (a) Human growth hormone, (b) Factor VIII, (c)  Pharmaceuticals
4.    Genetic Engineering in Yeast: Among Eukaryotes, yeast is commonly used in genetic engineering. Saccharomyces cerevisiae is termed as the E. coli of eukaryotes for the purpose of genetic engineering.
5.    Genetic Engineering in Plants :  
Example
i.    Herbicide-resistant plants
ii.    Cotton plants that are resistant to pests due to the incorporation of a bacterial gene that is toxic to insects
iii.    Soybeans that produce more healthy combinations of fatty acids
iv.    Flowers that stay fresher longer by inhibition of genes that are involved in senescence.
6.    Genetic Engineering in Aminals : 
i.    Basic research
a.    Knockout mice for determining the function of a gene
b.    Knockout mice for genetic disease models
ii.    Production of useful proteins (factor IX for treating haemophilia B)
iii.    Transgenic animals paved the way for gene therapy.
7.    Gene Therapy: Somatic gene therapy (introduction of the transgene into somatic tissues). Some diseases that have gene therapy studies in clinical trials (cystic fibrosis, muscular dystrophy, adenosine deaminase deficiency, familial hypercholesterolemia, cancer, AIDS).
8.    Screening for Genetic Diseases: Over 500 recessive diseases that have been identified by genetic engineering. For example, In Amniocentesis where Fetal cells are taken from the amniotic cavity via syringe and sampled for chromosome patterns, proteins, biochemical reactions.
9.    Forensic analysis: RFLP can be used in forensic testing. Results of the test are analyzed based on statistics, probability and population genetics.


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