Restriction Enzymes

By Mihir Kapse | 29 August 2024

Restriction enzymes, often called restriction endonucleases, are a class of enzymes (biological catalysts present in living organisms) that play a vital role in the defence mechanism of bacteria and archaea - and are used by synthetic biologists in Recombinant DNA technology.

The term ‘restriction enzyme’ originates from studying bacterial virus phage 𝜆 (the virus that attacks E. coli). They are thought to have likely evolved from a common ancestor and became widespread via horizontal gene transfer. The way they were discovered was basically through their functionality.

When a virus (specifically, a bacteriophage) attacks a bacterium, it inserts foreign DNA into the cell. Restriction enzymes cut this DNA into multiple fragments, making it less harmful.

How does the restriction enzyme know where it should cut? Each restriction enzyme is designed such that it recognises and cuts only a unique DNA sequence. This unique sequence is known as a recognition site. These are characterised by 4-8 bases which are generally a palindrome (Example: GTAATG) or a mirror-palindrome (DNA sequences where a segment of nucleotides read the same forward and backwards on both strands, Example: GATATC-CTATAG (complementary base pairing)). For example, the EcoRI (from E.coli bacteria) enzyme recognises the GAATTC base pair sequence, the Alul (from A. luteus) enzyme recognises the AGCT base pair sequence, and the Haell (from H. aegyptius) enzyme recognises the GGCC base pair sequence.

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There is the possibility that similar base pair sequences exist in the bacterial DNA also. Bacteria use the methylase enzyme to add methyl groups to their DNA to prevent the restriction enzyme from cutting its own DNA.

How do we use Restriction Enzymes? It has various applications.

  1. Recombinant DNA Technology: DNA fragments are cut from various sources using restriction enzymes and joined with other DNA sequences using DNA ligase to make new artificial DNA.
  2. Modular DNA Assembly: In processes like gene cloning and construction, restriction enzymes are used to assemble multiple DNA fragments in a predefined order.
  3. Site-Directed Mutagenesis: Scientists use restriction enzymes to make specific cuts and study the effects of these changes on gene function and regulations.

Presently, we know more than 3600 restriction enzymes from 250 different species. Researchers have been successful in studying over 3000 of these in detail and have commercialised 800 of these. We don’t know what the future holds for us. Someday, we might even be able to make artificial restriction enzymes that could be used for novel purposes, by manipulating the target site to suit our purposes. The possibilities are infinite!

References:

  1. Roberts RJ, Restriction and modification enzymes and their recognition sequences, Science Direct, Jan 1980
  2. Roberts RJ, Restriction endonucleases, CRC Critical Reviews in Biochemistry, 1976
  3. Roberts RJ, How restriction enzymes became the workhorses of molecular biology, Proceedings of the National Academy of Sciences of the United States of America, 2005
  4. Image: Microbe Notes

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