Restriction enzymes: scissors that read a sequence

Scissors that read

Imagine scissors that cut a long ribbon only where they find the word GAATTC written on it. They ignore the rest. Restriction enzymes behave like such scissors for DNA.

Where the enzymes come from

Bacteria are attacked by viruses called bacteriophages. A bacterium needs a defence. In 1963, two enzymes that restrict the growth of a bacteriophage in Escherichia coli were isolated. One of them added methyl groups to the DNA. The other cut the DNA. The second one is called a restriction endonuclease.

The first restriction endonuclease to be isolated was Hind II. It always cut DNA at a particular point, where it found a particular sequence of six base pairs. This sequence is its recognition sequence. NCERT says that more than 900 restriction enzymes have been isolated from more than 230 strains of bacteria, and they recognise different sequences.

Two kinds of nuclease

Restriction enzymes belong to a larger group, the nucleases. There are two kinds.

• Exonucleases remove nucleotides from the ends of the DNA.
• Endonucleases cut the DNA at specific positions inside it.

A restriction endonuclease moves along the DNA, and it "inspects" the sequence. When it finds its recognition sequence, it binds there. Then it cuts both strands of the double helix, in the sugar-phosphate backbone.

How an enzyme is named

The name tells where the enzyme came from. Take EcoRI.

The same rule gives HindIII (Haemophilus influenzae, strain d, the third enzyme) and BamHI (Bacillus amyloliquefaciens, strain H, the first enzyme). Only EcoRI is explained in NCERT, but the other two are standard examples in A-Level and IB books.

Palindromes

The recognition sequence of most of these enzymes is a palindrome. In ordinary words, a palindrome reads the same forwards and backwards, like "madam". In DNA, the meaning is a little different. A palindromic sequence has the same base order on the two strands, when both are read in the same direction, 5 prime to 3 prime.

EcoRI looks for this site.

• Top strand, 5 prime to 3 prime: GAATTC.
• Bottom strand, 5 prime to 3 prime: GAATTC.

To test any site, write the bottom strand by pairing the bases (A with T, G with C), and then read it backwards. If you get the same word, it is a palindrome.

Sticky ends and blunt ends

EcoRI does not cut in the middle of its site. It cuts between G and A, on both strands. Because the two strands are read in opposite directions, the two cuts are not opposite each other. The cuts leave a short stretch of single strand, four bases long, hanging from each piece. Here it is AATT.

These hanging stretches are called sticky ends. They are sticky because a single-stranded end can pair, by hydrogen bonds, with any other end that has a complementary sequence. The sticky ends made by one enzyme on any DNA match each other. This helps the enzyme DNA ligase in the next lesson.

Some enzymes cut straight through the middle of the site. They leave blunt ends, with no overhang. SmaI (CCC and GGG, cut between the C and the G in the middle) is an example. NCERT draws only sticky ends. Blunt ends are taught at A-Level and IB.

How often does a site appear?

A site of 4 bases can be written in 4 times 4 times 4 times 4, that is 256, different ways. So a given 4-base site turns up by chance about once in every 256 bases. A 6-base site turns up about once in every 4096 bases, and an 8-base site about once in 65536 bases. The longer the site, the rarer the cuts, and the larger the fragments.

Counting the fragments

• A straight piece of DNA with k cut sites breaks into k + 1 fragments.
• A circular DNA, such as a plasmid, with k cut sites (k at least 1) breaks into k fragments.

A plasmid with exactly one site is therefore turned into one straight piece. This is the reason why a good vector has just one site for an enzyme. Then the enzyme opens the plasmid and does not chop it up.

You should find that the average distance between sites grows very fast with the length of the site. Going from a 4-base site to a 6-base site makes the average distance 16 times greater.

Try it in Python

The program looks for the EcoRI site in a short piece of DNA and cuts the top strand after the G.

Practise with new numbers

Common mistakes

• Saying that a palindrome in DNA reads the same on one strand. It reads the same on the two strands in the same direction. On one strand, GAATTC read backwards is CTTAAG.
• Saying that restriction enzymes cut at random. Each cuts only at its own recognition sequence.
• Mixing up exonuclease and endonuclease. Exo works on the ends. Endo cuts inside.
• Using k + 1 for a plasmid. A circle with k cuts gives k pieces.
• Reading the Roman number as the number of cuts. The I in EcoRI means the first enzyme found in that strain.

Remember these

• A restriction endonuclease cuts both strands inside its palindromic recognition sequence.
• EcoRI: GAATTC, cuts between G and A, leaves the sticky end AATT. The name: Escherichia coli, strain R, the first enzyme.
• Exonucleases work at the ends of the DNA. Endonucleases cut inside.
• Straight DNA with k sites gives k + 1 fragments. A circular DNA with k sites gives k fragments.

Check yourself

Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.

1. Explain each letter in the name HindIII.

2. Is the sequence GGATCC a palindrome? Show how you know.

3. How is a sticky end different from a blunt end?

4. A straight DNA has 4 sites for an enzyme. A circular DNA has 4 sites for the same enzyme. How many fragments does each give?

5. About how far apart do you expect two chance sites of a 6-base enzyme to be?

References

• NCERT (2023). Biology, Textbook for Class XII, chapter 9, Biotechnology: Principles and Processes.
• Clark M. A., Douglas M. and Choi J. (2018). Biology 2e, OpenStax, chapter 17.2 (Basic techniques to manipulate genetic material).
• Roberts R. J. et al. (2003). A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Research 31, 1805 to 1812.