Restriction Enzymes

What are Restriction Enzymes?

Restriction enzymes are one of the biological tools that are used for the synthesis of recombinant DNA. Restriction enzymes are isolated from the bacteria and are protein in nature. They recognize a short, specific nucleotide sequence and cleave the DNA at that specific site, which is called a restriction site. When they recognize any specific nucleotide sequence in the DNA, they hydrolyze the bond between the adjacent nucleotides and cut the DNA molecule. Restriction enzymes are generally called restriction endonucleases because they cut within the DNA strands.

Types of Restriction Enzymes

They are classified into three types −

• Type I - These are complex, multi-subunit enzymes. They perform two functions: restriction and modification. They cleave the DNA approximately 1000 base pairs away from their recognition site. Therefore, they are not used in recombinant DNA technology and have very little practical value as they do not produce restriction fragments. Examples: EcoB, EcoK.

• Type II - These enzymes cleave the DNA within their recognition site and can be used in vitro for the production of recombinant molecules. Their recognition site is about 4-8 base pairs and causes cleavage at unmethylated sites. They require Mg²⁺ as their cofactor. Presently, more than 350 different type II restriction endonucleases with 100 different recognition sequences are known. Examples: EcoRI, BamHI, HindIII.

• Type III - They are complex enzymes and possess both the properties of restriction and methylation (modification). They cleave 25-27 base pairs away from their recognition site, so they are not used in recombinant DNA technology. Example: EcoPI, HinfIII.

Nomenclature

• The names of enzymes are derived from the names of the prokaryotic cells from which they are isolated.

• The first letter of the genus becomes the first letter of the name of the enzyme. It is written in capital letters.

• The first two letters of the species' name make up the second and third letters of the name of the enzyme. They are written in small letters.

• All these letters are written in italics.

• The fourth letter of the name of the enzyme is the first letter of strain. It is written in capitals.

• The order in which the enzymes were isolated from that particular strain of prokaryotic cell is indicated by the roman number at the end of the name.

Working Mechanism

Each restriction endonuclease inspects the DNA molecule in search of a specific recognition sequence. Once it gets the recognition sequence, it binds to the site and cuts each of the two strands of the DNA double helix at specific points by hydrolyzing the phosphodiester backbones.

The restriction sequence of each restriction nuclease consists of a definite number of specific base pairs in a DNA double helix. It is called a palindromic nucleotide sequence.

The cleavage of DNA strands by restriction enzymes can produce two types of fragments: fragments with sticky ends and fragments with blunt ends.

The fragments with sticky ends have an overhang because one fragment is longer than the other, while the blunt ends have straight cuts on both strands.

Importance of Studying Restriction Enzymes

The ability of the restriction enzyme to cleave the base pairs with high specificity makes it important to study restriction enzymes because of their wide use in Genetic Engineering and Molecular Biology techniques.

• Molecular Cloning - The restriction enzymes have been of utmost importance because of the synthesis of recombinant molecules. They are used to cut the gene of interest and the host DNA so that they can be incorporated together, leading to the formation of recombinant DNA molecules.

• DNA Mapping - The restriction enzymes are used to determine the structural information about the fragment of DNA. It is also called restriction mapping.

• Gene Sequencing - Restriction enzymes can be used to sequence a large molecule of DNA by digesting it and then placing it under a sequencer.

• Restriction Fragment Length Polymorphism- It involves the digestion of long sequences of DNA and then separating them on gel electrophoresis based on their length. These fragments were later transferred to a membrane containing labelled probes.

• Pulse Field Gel Electrophoresis - It involves the separation of large DNA fragments and has the ability to distinguish them from different strains of bacteria.

Current Trends

One of the methods still frequently employed by researchers to conduct DNA cloning experiments is restriction enzyme digestion. Today, researchers rely on restriction enzymes not only for their in-vitro experiments but also to perform virtually any process that involves analyzing, manipulating, and creating new combinations of DNA sequences. Presently, we can create artificial restriction enzymes with virtually any sequence specificity.

Conclusion

Restriction enzymes are an important biological tool that is frequently used in genomics. They are used to cut the DNA molecules at a specific site and to create a space in the host genome to insert the gene of interest, thereby creating the recombinant DNA molecule.

FAQs

Q1. How are bacteria immune to their own restriction enzymes?

Ans. The bacteria use the restriction and modification method to protect their own DNA from the action of restriction enzymes. Bacteria modify their restriction sites by adding methyl groups to the recognition sites.

Q2. Explain the vector.

Ans. The vector contains foreign DNA, it is a molecular vehicle to transport foreign DNA into the host cell. Vectors contain restriction sites (DNA is added) and marker site to identify the transformed cell.

Q3. What are the factors that affect the activity of restriction enzymes?

Ans. Ans: For proper restriction enzyme activity, factors that need to be controlled are-

• Star Activity - It is the alteration in the activity of restriction enzymes where they cleave at non-specific sites under suboptimal conditions. The factors that induce Methylated DNA: A number of DNA molecules have methyl groups added to their recognition sites, which makes them more resistant to being cut by specific restriction enzymes.

• Temperature - Most of the endonucleases optimally work at 37-degree Celsius. But some endonucleases' optimum temperature can be below 25 or greater than 65 degrees Celsius.

Q4. What are palindromic sequences?

Ans. The palindromes are a group of letters that form the same words when read either in a forward or backward direction. Similarly, the palindromic nucleotide sequence in the DNA molecule is the sequence of base pairs that read the same on both the strands (5’ to 3’) which lies in opposite direction.

5’- GAATTC - 3’

3’- CTTAAG - 5’

Q5. What do you mean by restriction mapping?

Ans. Restriction mapping, also called gene mapping, is used to determine the sequence of unknown segments by breaking them into pieces and identifying the locations of the breakpoints. After the segment is broken down by the restriction enzymes, they are examined by gel electrophoresis and are separated on the basis of their size.