Where Restriction Enzymes Came From

Recombinant DNA technology can be carried out only with a set of key tools — restriction enzymes, polymerase enzymes, ligases, vectors and a host organism. The first of these, and in many ways the most defining, is the restriction enzyme.

In 1963, two enzymes were isolated that were responsible for restricting the growth of a bacteriophage in Escherichia coli. One of them added methyl groups to DNA, protecting the host's own DNA, while the other cut the DNA. It was this second, DNA-cutting enzyme that came to be called a restriction endonuclease.

Hind II and the Recognition Sequence

The first restriction endonuclease whose action depended on a specific DNA sequence was Hind II, isolated and characterised about five years later. It was found that Hind II always cuts a DNA molecule at a particular point, because it recognises a specific sequence of six base pairs. This specific base sequence is known as its recognition sequence.

Hind II was only the beginning. Today we know of more than 900 restriction enzymes, isolated from over 230 strains of bacteria, and each one recognises a different recognition sequence. This diversity is what lets us cut DNA in so many precise, chosen ways.

How Restriction Enzymes Are Named

The names of these enzymes follow a simple convention that tells you where each one came from. The first letter is taken from the genus of the source organism, and the next two letters come from its species.

For example, EcoRI comes from Escherichia coli RY13. Here 'E' is from Escherichia, 'co' from coli, and the letter 'R' is derived from the name of the strain, RY13. The Roman numeral at the end indicates the order in which the enzymes were isolated from that particular strain of bacteria. So the very name of an enzyme records its genus, species, strain and sequence of discovery.

Nucleases and How the Enzyme Cuts

Restriction enzymes belong to a larger class of enzymes called nucleases, which come in two kinds. Exonucleases remove nucleotides from the ends of a DNA molecule. Endonucleases, by contrast, make cuts at specific positions within the DNA — and it is to this second group that restriction enzymes belong.

A restriction endonuclease works by first 'inspecting' the length of a DNA sequence. Once it finds its specific recognition sequence, it binds to the DNA and cuts both strands of the double helix at specific points in their sugar-phosphate backbones. This clean, sequence-specific cutting is precisely what makes these enzymes the molecular scissors of genetic engineering.

Quick Recap

  • In 1963, two enzymes that restrict bacteriophage growth in E. coli were isolated — one added methyl groups to DNA, the other cut DNA (the restriction endonuclease).
  • Hind II, characterised five years later, always cuts DNA by recognising a specific six-base-pair recognition sequence.
  • Today more than 900 restriction enzymes are known, from over 230 strains of bacteria, each recognising a different sequence.
  • Naming: first letter = genus, next two letters = species, e.g. EcoRI from Escherichia coli RY13 ('R' from the strain); Roman numerals show the order of isolation.
  • Restriction enzymes are nucleases: exonucleases cut from the ends, endonucleases cut at specific internal sites.
  • A restriction endonuclease inspects the DNA, binds at its recognition site and cuts both strands in their sugar-phosphate backbones.

Solved Examples — Section 4

Q1. In 1963, two enzymes restricting bacteriophage growth in E. coli were isolated. What did each do?

Answer: One added methyl groups to DNA, while the other cut DNA; the DNA-cutting one was the restriction endonuclease.

Q2. Name the first restriction endonuclease and state the length of its recognition sequence.

Answer: Hind II; it recognises a specific sequence of six base pairs.

Q3. How many restriction enzymes are known today, and from how many bacterial strains?

Answer: More than 900 restriction enzymes, isolated from over 230 strains of bacteria.

Q4. What does each part of the name EcoRI stand for?

Answer: 'E' is the genus Escherichia, 'co' the species coli, 'R' the strain RY13, and the Roman numeral I the order of isolation.

Q5. Distinguish between exonucleases and endonucleases.

Answer: Exonucleases remove nucleotides from the ends of the DNA, whereas endonucleases make cuts at specific positions within the DNA.

Q6. Once a restriction endonuclease finds its recognition sequence, what exactly does it cut?

Answer: It binds the DNA and cuts both strands of the double helix at specific points in their sugar-phosphate backbones.