Biotechnology and Bioinformatics (1+1)

2.1.2. Enzymes commonly used in recombinant DNA technology

2.1.1. Restriction endonuclease s (RE)

Restriction endonucleases are bacterial enzymes that are hydrolases and cleave phosphodiester bonds of double stranded DNA at specific palindromic sites within the chain to produce 5¹ PO₄ and 3¹ OH ends.

They are called as “restriction endonucleases” mainly due to their natural function in restricting the growth of the virus that attack bacteria. The enzymes do this by binding to the viral DNA and cleaving it at highly specific rites within or adjacent to a particular sequence know as recognition sequence.

The site specificity is important since it enables the bacteria to defend its own DNA against attack by the restriction enzyme s by methylating the corresponding sites of its own DNA. Thus each types of bacterium produces a few restriction endonucleases, but its own DNA is not cleaved by its own restriction enzyme as it is methylated and thus protected from cleavage by its own enzymes. The existence of restriction enzymes were observed by Werner Arber (1968).

About hundreds of different restriction enzymes have been purified, and many of them are commercially available. These restriction enzymes are utilized to cut any extremely long length of DNA into a series of appropriate sized fragments, from which a fragment containing the desired gene is probe d.

There are three types of restriction endonucleases : Type I, II and III.

Type I and III recognize specific nonpalindromic sequences in the DNA chain, but cleave the chain at different sites away from the recognition site, thus producing DNA fragments of different length and ends.

Type II restriction endonucleases, however, recognize specific palindromic sequences that range generally from 4–8 nucleotide s and cut the chain within the site, thus producing specific DNA fragments with known ends. For this reason, they are very useful in genetic engineering .

Palindrome s in DNA are sequences that read the same sequence of bases from either end (e.g. MALAYALAM)

The restriction enzyme ECO RI, recognizes the GAATTC sequence and cuts the two strands at the sites shown producing staggered or sticky or cohesive 5¹ and 3¹ ends. Alu I cuts the AGCT sequence producing blunt or flush ends.

At present more than 1200 restriction enzymes with different specificities for different palindromic sequences have been purified from various types of bacteria.

Certain restriction endonucleases purified from different bacteria recognize the same palindromic sequence, but may or may not cut at the same site and are named isoschizomers,

e.g. Dpn I and Sau 3A cut at GATC and G^mATC.

Hpa II cuts CCGG, but cannot cut C^mCGG.

Msp I cuts both CCGG and C^mCGG. Such isoschizomers are used for finding out if a DNA is methylated at a specific site.

The restriction enzymes are named by taking the first letter of the generic name and the first two letters of the species, e.g. the Eco RI. E. coli R – strain , I is the first enzyme. Specific site – GAATTC – Eco RII – Second enzyme, specific site – CCAGC -.

The frequency of occurrence of a palindrome in a DNA strand depends on the length of the palindrome. Site frequency = ¼ n where, n = length of the restriction site sequence. For example, an AGCT palindrome occurs at intervals of 256 bp, whereas GAATTC occurs at intervals of 4096 bp.

Restriction enzymes have helped in the isolation of specific genes from various species including that of man and transferring it to another species, and thus cross the species barrier, though in nature, two different species do not cross-breed. They have helped in the development of recombinant DNA and genetic engineering technology which are of immense benefit for mankind.