Restriction Enzyme

Restriction enzyme refers to as “Restriction endonuclease” which was discovered during the study of Entero-bacteriophage where the E.coli inhibits the phage activity. In 1978, Werner Arber, Daniel Nathans, Hamilton O Smith won the Nobel Prize for the characterization and discovery of restriction enzyme.

Restriction endonuclease also refers to as “Molecular scissors” which are widely used in the recombinant DNA technology or in the field of molecular biology to cut the foreign DNA. The enzyme catalyses the different reactions, and the restriction enzyme restricts the function of cleavage. Basically, these work as “Endonucleases” and hence also called “Restriction endonuclease”.

Content: Restriction Enzyme

  1. Definition of Restriction Enzyme
  2. Mechanism
  3. Modified Restriction Enzyme
  4. Nomenclature
  5. Genes for the Restriction-Modification System
  6. Genes for the Modification System only
  7. Types
  8. Working

Definition of Restriction Enzyme

A restriction enzyme is a type of endonuclease enzyme which functions to cleave the nucleotide sequences in between the DNA strand but the site of cleavage is specific for the restriction endonuclease.

In the DNA, there are some specific sequences are present termed as “Recognition or Restriction sequences”. The restriction endonuclease will recognize the restriction sequences and bind to the site and cleave the strand particularly on that site and breaks the DNA strand into two or more strands.

A restriction endonuclease can perform three functions like recognition of restriction site, cleavage in the restriction site and modification of DNA. The restriction endonuclease can be isolated from the bacteria and can be used in genetic engineering and cloning methods etc.

Mechanism

Let us suppose a bacterial cell infected by phage particle. Then we will see that the phage genome will enter into the bacterial genome. Then a war begins between a genome of both bacteria and the phage. Both will produce a restriction endonuclease as a weapon to degrade each other.

The bacterial genome will produce restriction enzyme for the degeneration of the phage DNA so that it could not take up the cell machinery. And, the phage DNA will produce a restriction endonuclease to integrate or to degrade the bacterial chromosome.

But there are recognition sites or sequences present on the bacterial DNA, which will produce restriction endonuclease. This restriction endonuclease cleaves the foreign DNA if that contains a particular sequence or will cleave the nucleotides of its own.

Modified Restriction Enzyme

It also refers as “Methylation enzyme”. Modified restriction endonuclease plays a crucial role in the recognition or differentiation of the bacterial vs foreign DNA. In the modified restriction endonuclease, the recognition sequences of the DNA are modified by the methylation.

Let us suppose, there is a recognition sequence of “GATC”, then the CH3 or methyl group will attach to the adenine and cytosine and thus modifying the bases. The modification of the bases guides the restriction endonuclease not to cleave the DNA of its own strand.

Modified restriction endonuclease is absent in the viral genomic DNA. This enzyme can either work independently or coordinate with the restriction endonuclease. Therefore, to prevent the cleavage of its own DNA, the bacteria will produce modifying restriction endonuclease which function is not only to cleave but also to modify its own DNA sequences by the process of methylation.

Nomenclature

First, the restriction enzyme is isolated from the bacteria and the nomenclature system depends upon the type of bacteria from which the enzyme has been isolated. To know the nomenclature system let us take some examples.

Example 1: Eco R-I

The first letter is always written in the capital where ‘E’ represents the “Genus” of the bacteria from which the restriction endonuclease has been isolated which is Escherichia. The second two letters represent the species of the bacteria which is coli written as ‘co’. The third letter i.e. ‘R’ represents the strain of the bacteria which varies with different species. The last is the number i.e. ‘I’ which signifies the order of identification of the enzyme. The recognition sequence of Eco R-I is 5’-GAATTC-3’.

Example 2: Bam H-I

Similarly, the letter ‘B’ represents the genus which is “Bacillus”. The letters ‘am’ represents the species which is amyloliquefaciens. And, the last two represents the strain number and the order of identification of the restriction endonuclease. The recognition sequence of Bam H-I is 5’-GGATCC-3’.

Genes for the Restriction-Modification System

The genes for the restriction modification system was first discovered in the E.coli of strain K-12. After the discovery, the genes which are involved in the modification system of restriction endonuclease named as “hsd”. The hsd stands for “Host-specific defence or Host specificity determinant”. The hsd genes in the E.coli of strain K-12, performs three functions, according to which it classifies into three types:

hsd-S: This gene includes the specificity factors i.e. restriction site or recognition sequences that help the restriction endonuclease to recognize and bind with the specific site of the DNA sequence.

hsd-M: This gene includes the modification factors which modify the nucleotide bases of the DNA by adding CH3 group which prevent the chopping up of its own DNA by the restriction endonuclease.

hsd-R: This gene codes for the particular site or sequences in the DNA, where the restriction endonuclease binds and cleave the strand into two or more fragments.

Genes for the Modification System only

The modified restriction enzyme can also work independently. The genes which control the modified restriction enzyme are of three types:

dam gene: Here dam stands for “DNA Adenine Methyltransferase”. The dam gene recognizes particularly 5’-GATC-3’ sequence and the methyl group attaches to the adenine base.

dcm gene: Here dcm stands for “DNA Cytosine Methyltransferase”. The dcm gene recognizes particularly 5’-CCATGG-3’ sequence and the methyl group attaches to the cytosine base.

mcr gene: Here mcr stands for “Modified Cytosine Restriction”. The mcr genes are of two types namely mcr B and mcr C. It recognizes the modified sequences and cut the DNA from the internal cytosine residue.

Types

The restriction endonuclease is mainly of three types namely type-I, II and III based on the following factors like enzyme system complexity, cofactor requirements etc.

On below, there is a comparison table of all the three types of restriction enzymes:

Properties Type-IType-IIType-III
Protein structureMulti-subunit complexMulti-enzyme complexMulti-subunit complex
Cleavage site1000bp downstream
At the recognition site
24-26bp downstream
Requirements of DNA cleavageTwo recognition sequences, in any orientationOne recognition sequenceTwo recognition sequences, in and out direction
Cofactors requirementATP, Mg2+ and sodium adenosyl methionineMg2+ATP, Mg2+ and sodium adenosyl methionine
Site of methylationAt the recognition siteAt the recognition siteAt the recognition site
Importance in cloningNot preferred for the cloningPreferred enzyme for the cloning and genetic engineeringNot preferred for the cloning

Type-I Restriction endonuclease: The type-I restriction endonuclease is a single enzyme but acts as a “Multi-subunit complex”. This enzyme creates a cleavage at 1000bp downstream to the DNA strand. Type-I restriction endonuclease requires two recognition sequences in any orientation. For the cleavage, it requires ATP, Mg2+ and sodium adenosylmethionine. In the process of cloning, it is not preferred because of the non-specificity of the cleavage site.

Type-II Restriction endonuclease: The type-II restriction endonuclease is a set of an enzyme that acts as a “Multienzyme complex”. This enzyme creates a cleavage specifically on the recognition site of the DNA strand. Type-II restriction endonuclease requires one recognition sequence. For the cleavage, it requires Mg2+. In the process of cloning, it is the most preferred enzyme because of its specificity in the cleavage site.

Type-III Restriction endonuclease: The type-III restriction endonuclease is a single enzyme with a “Multi-subunit complex”. This enzyme creates a cleavage at 24-26bp downstream to the DNA strand. Type-I restriction endonuclease requires two recognition sequences to read the DNA strand in the bi-directional movement facing towards each other. For the cleavage, it requires ATP, Mg2+ and sodium adenosylmethionine. In the process of cloning, it is not preferred because of the non-specificity of the cleavage site where one has to count the 26bp downstream to cut the DNA strand. The site of methylation occurs at the recognition site.


Working

As the restriction endonuclease can perform either of the two functions like cleavage or modification. Both the processes cannot occur simultaneously so a restriction enzyme has to decide when it has to modify or to cleave the DNA strand.

Case-1:  In bacterial DNA

When the bacterial DNA is having fully methylated DNA strand, then neither cleavage nor modification occurs by the restriction endonuclease and the modified restriction enzyme. Cleavage will not occur in this case because both the strands of DNA are methylated so it will not allow restriction endonuclease to cut its own strand. As the DNA is fully methylated, therefore the modified restriction enzyme cannot do any modifications.

But in case of hemimethylated DNA, only one strand of the DNA is methylated. Fully methylated DNA when replicate it will produce hemimethylated DNA. Therefore, in hemimethylated DNA, new strands of non-methylated DNA will produce. Therefore modified restriction endonuclease can modify the new strands of the hemimethylated DNA and the restriction endonuclease will not work in the methylated DNA strand.

Case-2: In phage DNA

In foreign cell, the DNA is non-methylated. In this case, the modified restriction enzyme will not work because the modification system is not found in the foreign cell. As there is no modification or methylation, the restriction endonuclease will cut the DNA strands into some fragments.

Leave a Comment

Your email address will not be published. Required fields are marked *