RESTRICTION ENZYMES

RESTRICTION ENZYMES

A restriction enzyme is a nuclease enzyme that cleaves DNA sequence at a random or specific recognition sites known as restriction sites. In bacteria, restriction enzymes form a combined system (restriction + modification system) with modification enzymes that methylate the bacterial DNA. Methylation of bacterial DNA at the recognition sequence typically protects the own DNA of the bacteria from being cleaved by restriction enzyme

There are two different kinds of restriction enzymes:

 (1) Exonucleases catalyses hydrolysis of terminal nucleotides from the end of DNA or RNA molecule either 5’to 3’ direction or 3’ to 5’ direction. Example: exonuclease I, exonuclease II etc. 

(2) Endonucleases can recognize specific base sequence (restriction site) within DNA or RNA molecule and cleave internal phosphodiester bonds within a DNA molecule. Example: EcoRI, Hind III, BamHI etc

EXONUCLEASES AND IT TYPES: 

EXONUCLEASE III: 

• Exonuclease III is a globular enzyme which has 3’→5’ exonuclease activity in a double stranded DNA. 
• The template DNA should be double stranded and the enzyme does not cleave single stranded DNA. 
• The enzyme shows optimal activity with blunt ended sequences or sequences with 5’ overhang. 
• Exonuclease III enzyme has a bound divalent cation which is essential for enzyme activity.
• The mechanism of the enzyme can be affected by variation in temperature, monovalent ion concentration in the reaction buffer, and structure and concentration of 3’termini. 
• The enzyme shows optimal activity at 37°C at pH 8.0.

MUNG BEAN NUCLEASES:

• As the name suggest, this nuclease enzyme is isolated from mung bean sprouts (Vigna radiata). 
• Mung bean nuclease enzymes can degrade single stranded DNA as well RNA. Under high enzyme concentration, they can degrade double stranded DNA, RNA or even DNA/RNA hybrids.
•  Mung bean nuclease can cleave single stranded DNA or RNA to produce 5’-phosphoryl mono and oligonucleotides.
• It requires Zn2+ ion for its activity and shows optimal activity at 37°C. 
• The enzyme works in low salt concentration (25mM ammonium acetate) and acidic pH (pH 5.0). 
• Treatment with EDTA or SDS results in irreversible inactivation of the enzyme. Mung bean nuclease is less robust than S1 nuclease and easier to handle. It has been used to create blunt end DNA by cleaving protruding ends from 5’ ends. This enzyme cannot produce nicks in a double stranded DNA but at higher concentration, it can generate nicks and cleave double stranded DNA.

RNases: 

Ribonucleases are a class of hydrolytic enzymes that catalyzes both the in vivo and in vitro degradation of ribonucleic acid (RNA) molecules into smaller components. Ribonucleases are classified into two types, Exoribonucleases and endoribonucleases. 

Exoribonucleases: The exoribonuclease is an exonuclease ribonuclease that degrades RNA by removing terminal nucleotides from either the 5′ end or the 3′ end of the RNA molecule.

Endoribonucleases: The endonuclease ribonuclease cleaves RNA molecules internally. It can cleave either single-stranded or double-stranded RNA, depending on the enzyme.

RNases I:

RNases A:

RNases H:

T4 PHOSPHO NUCLEOTIDE KINASE:

Polynucleotide kinases (PNK) are one such DNA modification enzymes that add phosphate groups to nucleic acid molecules. They transfer the gamma phosphate group from adenosine triphosphate (ATP) to the 5’ hydroxyl termini of DNA or RNA.

                        ATP + 5'-dephospho-DNA ${\displaystyle \rightleftharpoons }$ ADP + 5'-phospho-DNA

Thus, the two substrates of this enzyme are ATP and 5'-dephospho-DNA, whereas its two products are ADP and 5'-phospho-DNA. Polynucleotide kinase is a T7 bacteriophage (or T4 bacteriophage) enzyme that catalyzes the transfer of a gamma-phosphate from ATP to the free hydroxyl end of the 5' DNA or RNA. The resulting product could be used to end-label DNA or RNA, or in ligation reactions.

DUAL ROLE OF T4 Polynucleotide Kinase

The dual roles—kinase and phosphatase—performed by the enzyme reside in its separate domains. The N-terminal is a 5′-kinase domain with a nucleotide-binding motif GXXXXGK(S/T) and a C-terminal is a 3′-phosphohydrolase domain.

TYPES OF RESTRICTION ENZYMES

Restriction enzymes are traditionally classified into four types on the basis of subunit composition, cleavage position, sequence specificity and cofactor requirements. However, amino acid sequencing has uncovered extraordinary variety among restriction enzymes and revealed that at the molecular level, there are many more than four different types.

TYPE 1

Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but this type is not used as like as Type 2. 

Enzyme Activity:

Type I R–M enzymes are pentameric proteins of composition 2R+2M+S. They require ATP, Mg2+ and S-adenosylmethionine (SAM) for activity and display both REase and MTase activities. A trimer of 2M+S acts solely as an MTase