Enzymes in Recombinant DNA Technology
Different kinds of specific enzymes are used in genetic engineering (recombinant DNA technology). These include lysing enzymes, cleaving enzymes, synthesizing enzymes, joining enzymes and alkaline phosphatase.
• The palindromes in DNA are base pair sequences that are the same, when read forward (left to right) or backward (right to left), from a central axis of symmetry.
• For example, the following sequence reads the same on the two strands, in both 5′ -» 3′direction and in the 3′ -» 5′ direction.
• Restriction endonuclease EcoRI found in the colon bacteria E.coli, recognizes the base sequence GAATTC in DNA duplex, and cuts the DNA strands between G and A only, as shown below:
Types of restriction endonucleases
• Three main types of restriction endonucleases are type I, type II and type III.
• Type I Restriction Endonucleases consist of 3 different subunits. They require ATP, Mg2- and S-adenosyl-methionine for restriction. Type I restriction endonucleases recognize specific sites within the DNA, but they do not cut these sites.
• Type II Restriction Endonucleases are simple,and require Mg2+ ions for restriction. Only type II restriction enzymes are used in recombinant DNA technology, because they can be used in vitro, to recognize and cut within specific DNA sequence, typically consisting of 4 to 8 nucleotides, and cause cleavage at unmethylated sites within their recognition.
• Type III Restriction Endonucleases are intermediate between type I and type II. They possess both the activities of restriction as well as methylation. Therefore, they are not used in recombinant DNA technology.
Restriction modification system
• The restriction modification system consists of restriction endonucleases and a modification enzyme.
• These enzymes are present in many bacteria where they function as a part of their defence mechanism called the restriction modification system.
• A restriction enzyme (restriction endonuclease) identifies the introduced foreign DNA. The term restriction’ refers to the function of these enzymes in restricting the propagation of foreign DNA of bacteriophages in the host bacterium.
• The modification enzyme adds a methyl group to one or two bases usually ‘within’ the sequence recognised by the restriction enzyme.
• Once a base in a DNA sequence is modified by addition of a methyl group, the restriction enzymes fail to recognize and could not cut that DNA.
• This is how a bacterium modifies and therefore, protects its own chromosomal DNA from cleavage by these restriction enzymes.
• The first restriction endonuclease was HindW (hin- dee-two). Its functioning depends on a specific DNA nucleotide sequence. It was isolated from Haemophilus influenzae Rd.
• HindW always cuts DNA molecules at a particular point by recognising a specific sequence of six base pairs called recognition sequence and producing blunt ends.
• The restriction enzymes cut the strand of DNA, a little away from the centre of the palindrome sites, but between the same two bases of the opposite strands. This generates protruding 3’ or 5’ ends, i.e., one strand of the double helix extends some bases beyond the other.
• Due to the palindromic (symmetrical) nature of the target sites, the two protruding ends have complementary base sequence. As a result, they can readily pair with other complementary bases of the introduced foreign DNA sequence, by forming hydrogen bonds due to the action of enzyme DNA ligase. Hence, such ends are called cohesive or sticky ends.
• Some restriction enzymes cut both the strands of a DNA molecule at the same site so that the resulting ends have blunt or flush ends in which the two strands end at the same point.
• The sticky ends facilitate the action of the enzyme DNA ligase.
• Restriction endonucleases are used in genetic engineering to form ‘recombinant’ molecules of DNA, which are composed of DNA from different sources/ genomes.
Nomenclature of restriction enzymes
• Specific name (nomenclature) of the restriction enzyme is derived from the name of the source, such as bacteria e.g., HindII enzyme has been isolated from Haemophilus influenzae.
• The first letter of the bacterium’s genus becomes the first letter in the name of enzyme, which is written in capital letter, in italics.
• The first two letters of the species become the second and third letters in the name of the restriction enzyme, which are written in small letters, also in italics.
• The fourth letter in the name of the enzyme is the first letter of the strain, from which the enzyme was isolated and is written in capital letter.
• The Roman numeral written at the end of the name, indicates the order, in which the enzyme was isolated, from that strain of the bacterial cell.
Other enzymes used in rDNA technology
(i) Synthesizing enzymes: They play a role in the synthesis of DNA strands on suitable templates. They are further of 2 types:
• Reverse transcriptases, which help in the synthesis of complementary DNA strands by using mRNAs as a template.
• DNA polymerases which aid in the synthesis of complementary DNA strands on DNA template.
(ii) Joining enzymes, DNA ligases or sealing enzymes help in sealing gaps in DNA fragments.
• The enzymes, used most often in the rDNA technology is T4 DNA ligase.
• These act as a molecular glue. They join DNA fragments by forming phosphodiester bonds.
(iii) Alkaline phosphatase cut off phosphate group from the 5′ end of linearised circular DNA to check its recircularization.
• If this phosphate group is removed, this DNA cannot be ligated.