The enzymes fall under four general classes: (1) Nucleases, (2) Polymerases, (3) Lipases and (4) DNA end modifying enzymes.
Nucleases are nucleic acid degrading enzymes. These break phosphodiester bonds holding the nucleotides together. The nucleases are classified from different viewpoints. Some are nucleic acid specific and some others are single or double strand specific. RNA nucleases are ribonucleases and those of DNA are deoxyribonucleases. These are also classified as exonucleases and endonucleases.
Exonuclases degrade nucleic acids from their free termini by removing nucleoside monophosphates. Therefore, these enzymes are specific for linear DNA / RNA molecules.
These cannot act on circular DNA molecule having no free termini. Exonucleases are again of two types, based on the degradation from the3′ or 5′ end. An exonuclease that removes nucleotides from the 3′ end is known as ego (3′ -> 5′) and the one that removes from the 5′ end is ego (51 -> 3′).
(b) End nuclease:
Conversely, endonucleases do not require free termini for action. They make single stranded or double stranded cuts within the DNA / RNA strands. They can act on linear as well as circular DNA molecules.
Their action forms polynucleotide fragments of variable sizes. Most important, among endonucleases, are restriction endonucleases/ restriction enzymes (herein after referred to as RE).
Restriction End nuclease: These are specific enzymes, which recognize specific sequence called recognition sequences and make double stranded cuts either within the recognition sequence or at a variable distance from the recognition sequence. Therefore, it is logical to reef these enzymes to as molecular scissors. The point of cleavage is known as a restriction site there will be as many restriction sites as the number of recognition sites.
Nomenclature: REs are named by a three-alphabet abbreviation after the name of the bacteria species from which they are isolated.
The three alphabets are followed by the strain of the bacterium. In some REs, roman numerals like I, II, etc. follow to identify multiple REs isolate from the same species. The first enzyme is designated by the Roman numeral, I and the subsequent enzymes by II and III and so on. For example, Eco RI is the first RE isolated from RY13 star Of Escherichia coli.
The subsequent enzyme from the same species and same strain is name as Eco RII. Around 500 REs have, so far, been isolated from bacteria. A few other RES commonly used in the cutting of the DNA are Him d III, Bam HI, PST I and Hap I.
Mechanism of Cutting: Most of the REs are specific for short sequences, called palindrome and cleave phosphodiester bonds on both the strands generating 3′-OH and 5′-I ends; palindrome is a sequence of base pairs, which reads the same in both the strands in the Sam direction. For example, the sentence “Madam, I am Adam” reads the same in the river direction.
This sentence is a palindrome. Some REs cleave both DNA strands symmetrical! Around the line of symmetry in the recognition sequence, yielding fragments with sticky cohesive ends. Such type of cutting is known as staggered cutting, cohesive end has an overhanging single stranded fragment, which is complementary to the oath cohesive end on the other strand.
A fragment having two cohesive ends circularizes spontaneous by complementary base pairing between two overhanging cohesive ends. Such circles linearity by heating.
A few other enzymes cleave both the strands on the line of symmetry oft recognition sequence yielding blunt/flush ended fragments. REs, which recognize the same recognition sequences, are termed as isoschizomers (Sau 3A and Mbo I). Thou isoschizomers recognize the same recognition sequence, they necessarily do not cleave at same position (Sam I and Exam I).
Polymerases are enzymes that copy nucleic acid molecules. They are primarily classified as (1) RNA polymerase / DNA dependent RNA polymerase and (2) DNA polymerase / DNA dependent DNA polymerase based on the substrate.
There is a third type of polymerase, the reverse transcriptase / RNA dependent DNA polymerase in retroviruses. An RNA polymerase catalyzes the copying of a strand of DNA into RNA through a process known as transcription.
DNA polymerases are enzymes that catalyze the copying of a DNA strand into another complementary DNA strand through a process known as replication. There are three DNA polymerases in prokaryotes: I, II and III.
(a) DNA Polymerase I:
It was believed to be the chief DNA polymerizing enzyme. However, later, it was found to have a proof reading function during replication. In addition to the polymerase function, it has 3′ -> 5′ and 5′ 3′ exonuclease functions.
The 3′ 5′ exonuclease function removes any impaired nucleotide during replication and thus increases the fidelity of the process. The 5′ -> exonuclease function lies on a different sub-unit of the enzyme. It can be remove by cleavage to result in a Keno fragment that retains the polymerase 3′ -> 5′ exonuclease functions.
The Keno fragment is used to copy a sin stranded DNA fragment. The 3′ 5′ exonuclease function is suppressed? Copying the single stranded DNA. The Keno fragment is used for radiolabel of probes by primed synthesis and in DNA sequencing by di-decoy meth The 5′ -> 3′ exonuclease function degrades the non-template strand sat polymerase synthesizes a complementary strand of the template strand. The procedure is known as nick translation for radio labeling of DNA.
(b) DNA polymerase II and III:
Polymerase II undertakes the major repair woo in the DNA and the polymerase III is the principal replicating enzyme.
(c) Reverse Transcriptase:
It is also called RNA dependent DNA polymer as it uses an RNA as the template to synthesize a complementary strand DNA. This single stranded DNA is called c (complementary) DNA. TO enzyme is used for copying mRNA into canal in gene manipulation procedure
3. DNA Lipases:
Lipases are cellular enzymes that seal or repair broken phosphodiest bonds in a sugar-phosphate backbone. In gene manipulation, the enzyme is used to se the discontinuities, which may arise when a recombinant DNA is made by joining t DNA fragments from two different sources. It can therefore, be thought of as molecu glue, which sticks pieces of DNA together. The enzyme used in gene manipulation experiments is the T4 DNA IGATE, which is purified from E. coli cells, infected by bacteriophages.
4. End Modifying Enzymes:
These enzymes modify the termini of DNA molecules in variety of ways, which are used in gene manipulation procedure in some way toroth Important ones among these are alkaline phosphates, polynucleotide kinas a terminal transferees. Alkaline phosphates removes phosphate groups from the end of DNA, leaving a 5′-OH group. It prevents unwanted ligation of DNA molecule
It is also used to remove the phosphate group before tagging a radio labeled phosphate group to the 5′-OH, catalyzed by the enzyme polynucleotide kinas. Terminal transferees adds homopolymeric tails (poly-A and poly-TO) to blunt ends of DNA so as to make the ends cohesive for complementary base pairing during recombinant DNA formation.