Different kinds of specific enzymes are employed in genetic engineering or recombinant DNA technology. Restriction enzyme, also called restriction endonuclease, is a protein produced by bacteria that cleaves DNA at specific sites along a molecule. These enzymes have been observed to cleave foreign DNA in bacterial cells, thus eliminating infecting organisms. They can be isolated from bacterial cells and used to manipulate fragments of DNA in the laboratory. Therefore, restriction endonucleases are considered to be pivotal in recombinant DNA technology/genetic engineering. Read on to learn more about these amazing enzymes.

Restriction Enzymes: Definition

Restriction enzyme, which is also known as Restriction Endonuclease, is a protein that is produced by bacteria that cleaves DNA at specific sites along the molecule.

Fig: Restriction Enzyme

These enzymes have the capacity to recognize the specific base sequences on the DNA helix and then cut each strand at a given place. Hence, they are also known as “molecular scissors“.

History

The first restriction enzyme was isolated and characterized as restriction enzyme (HindII) in the year \(1970.\) From then, over \(3000\) restriction enzymes have been studied in detail, and more than \(600\) of these are available commercially and are regularly used for DNA modification and manipulation in labs.

The two enzymes responsible for restricting the growth of bacteriophage in E. coli were isolated in \(1963.\) One was the enzyme that added methyl groups to DNA, while the other to cut the DNA. Later it was called restriction endonuclease.  Based on their mode of action, these enzymes are classified into Exonucleases and Endonucleases.

Exonucleases	Endonucleases
i. These nucleases cleave base pairs of DNA at their terminal ends.	i. These may be non-specific and make the cut at a random position within the DNA.
ii. They act on a single strand of DNA or gaps in double-stranded DNA.	ii. These enzymes do not cleave the ends and initiate only one strand of the DNA duplex.
iii. They do not cut RNA.	iii. They may cut RNA.

Mechanism

Restriction Endonucleases scan the length of the DNA and bind to the DNA molecule when it recognizes a specific sequence and later makes one cut in each of the sugar-phosphate backbones of the double helix of the DNA by hydrolyzing the phosphodiester bond.

Specifically, the bond between the \(3’O\) atom and the P atom is broken down, and \(3’OH\) and \(5’PO_4^{3 – }\) is produced. \(M{g^{2 + }}\) is necessary for the catalytic activity of the enzyme. The enzymes hold the water molecule in a position where it can attack the phosphoryl group and also help in polarizing the water molecule towards deprotonation.

Fig: Restriction Enzyme

What is a Palindrome Sequence?

The DNA sequences recognized by restriction Endonucleases are known as palindromes. Palindromes are the base sequences that read the same on the two strands but in opposite directions.
For Example – the following sequences read the same on the two strands in the \(5’\) to \(3’\) direction; this also holds if read in the \(3’\) to \(5’\) direction. The EcoRI cutting site is given as:
\(5′ – {\text{GAATTC}} – 3’\)
\(3′ – {\text{CTTAAG}} – 5’\)

i. The mirror-like palindrome in which the same forward and the backwards are on a single strand of DNA strand, as in GTAATG.
ii. The inverted repeat palindromes are also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (GTATAC being complementary to CATATG).
iii. Inverted repeat palindromes are most common and have greater biological importance than mirror-like palindromes.

Ends of Restriction Fragments

These Restriction fragments produce two types of cut ends, i.e., blunt ends and sticky ends.

1. Blunt Ends – Some restriction Endonucleases cut DNA at the opposite base that leaves blunt-ended DNA fragments. These blunt-ended fragments can be joined to any other DNA fragment with blunt ends only.

Fig: Blunt Ends

2. Sticky Ends/Cohesive Ends – Most restriction enzymes make staggered cuts, and these cuts produce single-stranded sticky ends. DNA fragments with complementary sticky ends can be combined to create new molecules, which allow the creation and manipulation of DNA sequences from different sources.

Fig: Sticky Ends

Type I Restriction Endonucleases

i. These type I restriction Endonucleases recognize specific sites within the DNA but do not cut these sites and cut the DNA far from the recognition sequence. Hence, these are not used in recombinant DNA technology.
ii. These consist of three subunits. They require \(M{g^{2 + }},\) ATP and S-adenosyl methionine for restriction.

Type II Restriction Endonucleases

i. These are the most commonly available and used restriction Endonucleases as they recognize and cut or cleave DNA at the same site.
ii. They are composed of only one subunit. Their recognition sites are usually palindromic and \(4-8\) nucleotides in length.
iii. They do not use ATP for their activity.
iv. They usually require only \(M{g^{2 + }}\) as a cofactor.
v. The sites of the cuts induced by type II restriction endonucleases are shown by arrows.
vi. Some of these enzymes cleave DNA molecules to give rise to blunt ends, while others give sticky or cohesive ends.
vii. Some common examples of Type II restriction endonucleases are BamHI, EcoRI, HindIII, SalI, etc.

Type III Restriction Endonucleases

i. This type is an intermediate between type I and type II. They possess both the activities of restriction as well as the methylation process.
ii. They cut DNA about \(20-30\) base pairs after the recognition site, and therefore, they are not used in recombinant DNA technology.

Type IV Restriction Endonuclease

i. Type IV enzymes recognize modified, typically methylated DNA targets.
ii. Examples include methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA.

Applications

Some of the applications are as follows:
i. Isolated restriction enzymes are used to manipulate DNA for different scientific applications.
ii. They are used to assist the insertion of genes into plasmid vectors during gene cloning and also in protein expression experiments.
iii. They can also be used to distinguish gene alleles by specifically recognizing single base changes in DNA as single nucleotide polymorphism.
iv. They are used in DNA fingerprinting techniques.

Summary

Our understanding of restriction enzymes has paved the way for several breakthroughs in the field of medicine, agriculture, etc. These modifying enzymes perform the cutting and joining operations in DNA manipulation and genetic engineering. They are tools for monitoring Restriction Fragment Length Polymorphisms (RFLP) and this enables us to locate the mutation, identify disease-causing genes, generate human linkage maps and also further develop DNA fingerprinting techniques.

Frequently Asked Questions (FAQs)

The most commonly asked questions about restriction endonuclease are answered here:

Q.1. What is a Restriction Enzyme? Ans: Restriction enzyme, which is also known as Restriction Endonuclease, is a protein that is produced by bacteria that cleaves DNA at specific sites along the molecule.

Q.2. Write two examples of Restriction endonucleases? Ans: The two examples of restriction endonucleases are EcoRI and BamHI.

Q.3. What is the source of restriction enzymes? Ans: Bacterial species or cells are the major sources of restriction enzymes.

Q.4. What is the difference between Type I and Type II restriction endonucleases? Ans: The Type I restriction endonuclease cleaves DNA at random sites far from its recognition sequence, but the Type II restriction endonuclease cleaves DNA at defined positions close to or within its recognition sequence.

Q.5. Give one application of Restriction enzymes. Ans: They are used to assist the insertion of genes into plasmid vectors during gene cloning and also in protein expression experiments.

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