Restriction Enzymes:

 

For Recombinant DNA technology we need to cut the DNA at specific sites, which are recognized and cleaved by specific enzymes, which are described as restriction enzymes.

In the year 1963, the two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated. One of these added methyl groups to DNA, while the other cut DNA. The later was called restriction endonuclease.

The first restriction endonuclease–Hind II, whose functioning depended on a specific DNA nucleotide sequence was isolated and characterised five years later. It was found that Hind II always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs. This specific base sequence is known as the recognition sequence for Hind II. Besides Hind II, today we know more than 900 restriction enzymes that have been isolated from over 230 strains of bacteria each of which recognise different recognition sequences.

Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two kinds; exonucleases and endonucleases.

Exonucleases : Exonucleases remove nucleotides from the ends of the DNA or RNA molecule i.e., they 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.

 Endonucleases: Endonucleases make cuts at specific positions within the DNA or RNA molecule i.e., they 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.

 Each restriction endonuclease functions by scrutinizing or inspecting’ the length of a DNA sequence”and make the cut at the specific site called the restriction site. Once it finds its restriction site or specific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar -phosphate backbones.

The exact nature of the cut or cleavage produced by restriction endonuclease is very important in creating a recombinant DNA. Cleavage by an endonuclease creates DNA sequence with either a sticky end or blunt end.

Many restriction endonucleases make a simple double stranded cut in the middle of the recognition sequence, resulting in a blunt or flush ends.

The blunt ended fragments can be joined to any other DNA fragment with blunt ends using linkers/adapters, making these enzymes useful for certain types of DNA cloning experiments.



 

 

 Other restriction endonucleases cut DNA in a slightly different way. With these enzymes the two DNA strands are not cut at exactly same position. Instead the cleavage is staggered, usually by two or four nucleotides, so that the resulting DNA fragments have short- single stranded overhangs at each ends. These are called sticky or cohesive ends, as base pairing between them can stick the DNA molecule back together again because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.

 One of the important feature of sticky-ends enzymes is that restriction endonucleases with different recognition sequences may produce the same sticky ends. BamHI (recognition sequence GGATCC) and BglIII (recognition sequence AGATCT)  are examples, both produce GATC sticky ends




 Production of these sticky ends is the second feature of restriction enzymes that makes them suitable for Recombination DNA technology. The principle is simple, that if two different DNA molecules are cut with the same restriction enzymes, both will produce fragments with the same complementary sticky ends, making it possible for DNA chimeras to form.

Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA. Pallindroms are groups of letters that form the same words when read both forward and backward, e.g., “MALAYALAM”.

As against a word-palindrome where the same word is read in both directions, the palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same. For example, the following sequences reads the same on the two strands in 5'     3' direction. This is also true if read in the   3'    5' direction. 5' —— GAATTC —— 3' , 3' —— CTTAAG —— 5'

Which means that both strands have the same nucleotide sequence but in anti-parallel orientation. The enzyme cuts within this sequence.

Restriction Enzyme Nomenclature:

Restriction endonucleases are named according to the organism in which they were discovered, using a system of letters and numbers. The first letter of the name comes from the genus and the second two letters come from the species of the prokaryotic cell from which they are isolated. For example, For example, the name EcoRI comes from Escherichia coli RY 13. E is the genus name Escherichia, co- two letters from the species name and  the letter ‘R’ is derived from the name of strain. Roman numbers following the names indicate the order in which the enzymes were isolated or discovered from that particular strain.

 Similarly, HindIII (pronounced “hindee-three”) was discovered in Haemophilus influenza (strain d).


Classification of Restriction Endonucleases:

There are three major classes of restriction endonucleases based on the types of sequences recognized, the nature of the cut made in the DNA, and the enzyme structure:

• Type I restriction enzymes

• Type II restriction enzymes

• Type III restriction enzymes

 

 Type I restriction enzymes:

These enzymes have both restriction and modification activities. Restriction depends upon the methylation status of the target DNA.

Cleavage occurs approximately 1000 bp away from the recognition site.

 The recognition site is asymmetrical and is composed of two specific portions in which one portion contain 3–4 nucleotides while another portion contain 4–5 nucleotides and both the parts are separated by a non-specific spacer of about 6–8 nucleotides.

They require S-adenosylmethionine (SAM), ATP, and magnesium ions (Mg2+) for activity.

These enzymes are composed of mainly three subunits, a specificity subunit that determines the DNA recognition site, a restriction subunit, and a modification subunit

 

 Type II restriction enzymes:

Restriction and modification are mediated by separate enzymes so it is possible to cleave DNA in the absence of modification. Although the two enzymes recognize the same target sequence, they can be purified separately from each other.

Cleavage of nucleotide sequence occurs at the restriction site.

These enzymes are used to recognize rotationally symmetrical sequence which is often referred as palindromic sequence.

These palindromic binding site may either be interrupted (e.g. BstEII recognizes the sequence 5´-GGTNACC-3´, where N can be any nucleotide) or continuous (e.g. KpnI recognizes the sequence 5´-GGTACC-3´).

They require only Mg2+ as a cofactor and ATP is not needed for their activity.

Type II endonucleases are widely used for mapping and reconstructing DNA in vitro because they recognize specific sites and cleave just at these sites.

 

Type III restriction enzymes:

These enzymes recognize and methylate the same DNA sequence but cleave 24–26 bp away.

They have two different subunits, in which one subunit (M) is responsible for recognition and modification of DNA sequence and other subunit (R) has nuclease action.

Mg+2 ions, ATP are needed for DNA cleavage and process of cleavage is stimulated by SAM.

Cleave only one strand. Two recognition sites in opposite orientation are necessary to break the DNA duplex.

 

Property

Type I RE

Type II RE

Type III RE

Abundance

Less common than Type II

Most common

Rare

Recognition site

Cut both strands at a non- specific location > 1000 bp away from recognition site

Cut both strands at a specific, usually palindromic recognition site (4-8 bp)

Cleavage of one strand, only 24-26 bp downstream of the 3´ recognition site

Restriction and

modification

Single

multifunctional

enzyme

Separate nuclease

and methylase

Separate enzymes

sharing a common subunit

Nuclease subunit

structure

Heterotrimer – Three subunits

 

Homodimer – One subunit

Heterodimer – Two subunits

Cofactors

ATP, Mg2+, SAM

Mg2+

Mg2+ (SAM)

DNA cleavage

requirements

 

Use in Recombinant DNA

Two recognition

sites in any

orientation

 

Not Useful

Single recognition

site

 

 

Useful

Two recognition

sites in a

head-to-head

orientation

 

Not Useful

 

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