Lesson Explainer: Using Restriction Enzymes Biology

In this explainer, we will learn how to explain the function of restriction enzymes and outline the purpose of sticky ends.

DNA itself is a massive molecule, and in humans, it is composed of billions of base pairs with tens of thousands of different genes. To study our genome efficiently, it would make a lot of sense to break it up into smaller sections that are easier to manage.

Restriction enzymes (also called restriction endonucleases) are like molecular scissors that can cut out specific sequences of DNA. Restriction enzymes can be used to cut out genes of interest to study them individually. This capability has led to the characterization of a massive number of genes and the development of new and exciting DNA technology. Recombinant DNA technology is the combination of DNA from two different sources to create new genetic information. Restriction enzymes are a critical part of this, and without them we would not have vaccines, insulin, or many of the foods we eat!

Definition: Restriction Enzyme (Restriction Endonuclease)

A restriction enzyme is an enzyme that cuts DNA at a specific sequence.

Definition: DNA (Deoxyribonucleic Acid)

DNA is the molecule that carries the genetic instructions for life. It is composed of two strands of deoxyribonucleotides that coil around each other to form a double helix.

Definition: Recombinant DNA

Recombinant DNA is the combination of DNA from at least two different sources to form new genetic information not previously found in the genome.

Restriction enzymes are found naturally in bacteria and are used as a defense against viral infection. When a virus infects a bacterium, it inserts its DNA into the cell in order to produce more copies of itself. In response to this, the bacterium can produce restriction enzymes to recognize this viral DNA and cut it into smaller pieces or fragments, which disrupts the genetic information contained in the viral DNA. This prevents the virus from continuing its life cycle, or “restricts” it from infecting more bacterial cells. Restriction enzymes literally save bacterial lives!

Example 1: Understanding Where Restriction Enzymes Come From

From what microorganisms are restriction enzymes most commonly obtained?

  1. Viruses
  2. Protists
  3. Algae
  4. Fungi
  5. Bacteria

Answer

Restriction enzymes are like molecular scissors that cut DNA into fragments. They were originally discovered in bacteria as a way for bacteria to defend themselves against viruses. Restriction enzymes can recognize free-floating DNA (like the DNA a virus would inject into the bacterial cell during infection) and cut the DNA into smaller pieces so it can be degraded. This prevents the virus from continuing its life cycle inside the bacterium.

Therefore, the correct answer is option E, bacteria. Restriction enzymes are not found in the other indicated organisms.

You may recall that enzymes are proteins that function as biological catalysts to speed up chemical reactions. Enzymes always have a substrate that they interact with specifically to produce some product. In the case of restriction enzymes, the substrate is double-stranded DNA and the product is cut DNA. Restriction enzymes can catalyze the hydrolysis of the phosphodiester bond that makes up the backbone of the DNA molecule, to “cut” the DNA and form fragments. You can see this in Figure 1.

There are many types of restriction enzymes, and each one has its own target DNA sequence, or recognition sequence, that the restriction enzyme specifically recognizes. In Figure 2, we will be looking at the restriction enzyme EcoRI (pronounced Eco-R-1), which recognizes the DNA sequence GAATTC.

Key Term: Recognition Sequence

A recognition sequence is the specific sequence of DNA that a restriction enzyme recognizes and cleaves.

EcoRI recognizes GAATTC on the top strand as well as the corresponding CTTAAG on the bottom strand. Here is an interesting observation—instead of reading the bottom sequence in Figure 2 from left to right, try reading it from right to left. So instead of “CTTAAG” it reads “GAATTC,” which is actually the same sequence as the top strand!

This property, where the sequence reads the same forward as it does backward, is called a palindrome. A common example of a palindrome is the word “radar”—it reads the same forward and backward! A palindromic sequence is how many restriction enzymes recognize their target sequence.

Key Term: Palindrome

A palindrome is a word or sequence that reads the same forward and backward (for example, “radar”). Palindromes are how restriction enzymes recognize their target sequences. They read the same when read in the 5 to 3 direction on both strands.

When we talk about a word like “radar,” it makes sense to describe reading the word left and right. But with DNA there is no “left” and no “right.” The two DNA strands run in opposite directions to each other, so one strand goes in one direction, while the other strand goes in the opposite direction. But what defines this direction?

By convention, we describe direction in DNA as the way a single DNA strand is synthesized. DNA is always synthesized in the 5 to 3 direction, referring to the carbons where new deoxyribonucleotides are added in a growing DNA strand. The carbons in a deoxyribonucleotide are numbered 1 to 5, as shown in Figure 3. When DNA is synthesized, the phosphate group (attached to the 5 carbon) of a new deoxyribonucleotide is added to the 3 carbon of the growing DNA strand.

Key Term: 5′ and 3′

By convention, DNA sequences are written in the 5 (five prime) to 3 (3 prime) direction, which is the same direction that DNA is synthesized in. These numbers refer to the specific carbon in the deoxyribose backbone of DNA to which a new deoxyribonucleotide bonds.

Now that we understand how directionality in DNA works, let’s look again at the palindromic sequence for EcoRI: 5335.-GAATTC--CTTAAG-

Looking at the 5 to 3 strand, we can see the sequence GAATTC, and if we look at the 3 to 5 strand in reverse (so that we are reading it from the 5 to 3 direction), it also reads GAATTC. As stated earlier, this is a palindrome, and this is how restriction enzymes can recognize their target sequence. Just like the palindromic word “radar” reads the same from the left and right, a palindromic DNA sequence reads the same on both strands in the 5 to 3 direction.

Example 2: Recognizing Palindromic Sequences

Some restriction enzymes recognize a section of DNA that is the same sequence read 5-3 on one strand as it is read 5-3 on the complementary strand. An example of this is shown in the diagram.

What term is given to this pattern?

  1. Palindromic
  2. Aligned
  3. Complementary
  4. Supercoiled
  5. Canonical

Answer

Restriction enzymes are like molecular scissors that cut DNA into fragments. They are able to recognize specific sequences of DNA that are called recognition sequences. Each restriction enzyme has its own unique recognition sequence. Many recognition sequences are palindromic. This means that the sequence is the same on both strands when read in the 5 to 3 direction.

5 and 3 refer to the orientation of the carbons in the deoxyribonucleotide. When DNA is synthesized, the phosphate group (attached to the 5 carbon) of a new deoxyribonucleotide is added to the 3 carbon of the growing DNA strand. This means that each strand of DNA is synthesized in opposite directions. By convention, and shown below on the right, DNA is written in the 5 to 3 direction on top with the opposing 3 to 5 strand on the bottom.

If you now look back at the question, you can see that the sequence GAATTC is GAATTC on both strands when read in the 5 to 3 direction.

Therefore, the correct answer is option A, palindromic.

GAATTC is the recognition sequence of the EcoRI restriction enzyme. This is a palindromic sequence and has a very specific shape that is recognized by the EcoRI. So, in a bacterium that has been infected with viral DNA, numerous EcoRI restriction enzymes will attach to the viral DNA and scan it until they find this sequence. The enzyme will bind tightly to its recognition sequence, once found, and cut the DNA strands into smaller fragments as shown in Figure 4.

Since the genetic instructions for the virus are located within its DNA, when the DNA is cut by a restriction enzyme, these instructions are lost and the virus can no longer continue its life cycle. Restriction enzymes can therefore protect the bacterium, and as of 2014, there has been over 4‎ ‎000 discovered restriction enzymes!

When restriction enzymes cut DNA at their recognition sequences, they can produce fragments of a specific size. For example, if a 1‎ ‎000-base-pair piece of DNA contains an EcoRI recognition site at the 250-base-pair section, then treatment with EcoRI will produce 2 fragments: one that is 750 base pairs and another that is 250 base pairs. These recognition sequences exist in DNA by chance. So, longer pieces of DNA are more likely to contain more recognition sequences than shorter pieces of DNA.

Since DNA is cut by restriction enzymes, you may be wondering how the host’s DNA is protected. DNA can be modified by methylation, where methyl groups are added to certain nucleotide sequences. This can change the shape of the recognition sequence and prevent restriction enzymes from interacting with it.

Example 3: Understanding the Action of Restriction Enzymes

Samples of DNA are taken from two organisms and mixed with restriction enzyme BamHI. The restriction enzyme cuts the DNA from organism A into three sections but cuts the DNA from organism B into only two. What does this suggest about the DNA of the organisms?

  1. Organism A has fewer BamHI recognition sequences in its DNA than organism B.
  2. Organism A has more BamHI recognition sequences in its DNA than organism B.
  3. The DNA of organism B is longer than that of organism A.
  4. The sample taken from organism B is not mixed with enough restriction enzymes.

Answer

BamHI is an example of a restriction enzyme. Restriction enzymes, or endonucleases, are enzymes that cut DNA molecules at specific recognition sites. Each restriction enzyme will have a different recognition sequence.

Let’s assume the DNA from both organisms is linear; it does not form a circle. Since BamHI cuts the DNA from organism A into 3 fragments, this suggests that the DNA from organism A has 2 recognition sequences for BamHI. And since cutting the DNA for organism B with BamHI produced 2 fragments, this suggests that the DNA for organism B has only 1 recognition sequence. Look at the following figure to visualize this.

Because these recognition sequences are random, we could argue that the DNA from organism A is longer than that for organism B. This is because a longer sequence of DNA would have more chances of including the BamHI recognition sequence. However, this is assuming that the DNA between organisms A and B has similar proportions of nucleotides A, G, C, and T. Since we are not given any additional information to give a definitive answer, option C is not the best choice.

Option D implies that there are not enough restriction enzymes to carry out the reaction. If there are not enough restriction enzymes, and the reaction is not given enough time to proceed, then this might be possible. However, the question is asking about the DNA of the organisms, not about the reaction conditions, so option D is incorrect.

Therefore, the correct answer is option B, Organism A has more BamHI recognition sequences in its DNA than organism B.

The function of restriction enzymes is all well and good for bacteria, but how does it benefit us humans? Well, it gives us a way to cut DNA—any DNA, not just viral DNA—at very specific regions that are interesting to us. And because there are so many restriction enzymes, chances are we can cut out just about any interesting gene that we want to study. Or, in the absence of recognition sequences, it is also possible to engineer them into DNA.

But all this cutting can only go so far. What is the fun in cutting if we cannot stitch genes back together in new and exciting ways? Lucky for us, restriction enzymes have another interesting quality—some of them can produce what we call “sticky ends.”

Restriction enzymes can cut DNA in two ways. Either by forming sticky ends or by forming blunt ends.

Restriction enzymes that cut to leave sticky ends are able to cut DNA in such a way that they leave overhangs of DNA that are “sticky” for one another because of their complementary base pairs, as shown in Figure 5. It is this affinity that the bases have for one another that makes them “sticky.”

Definition: Sticky Ends

The same restriction enzymes can cut DNA to produce “sticky ends,” which are unpaired DNA sequences that can easily base pair with the complementary sequence on the other strand. For example, 5335.-GATCTGACTGATGC--CTAGACTGACTACG-

Example 4: Recognizing Sticky Ends

The diagram provided shows a fragment of DNA produced from the cutting of a sequence using BamHI. The fragment is left with exposed nucleotide bases. What term is given to this?

  1. Free ends
  2. Active ends
  3. Sticky ends
  4. Open ends
  5. Blunt ends

Answer

Restriction enzymes are like molecular scissors that cut DNA into fragments. They are able to recognize specific sequences of DNA that are called recognition sequences. Each restriction enzyme has its own unique recognition sequence. In this example, the restriction enzyme BamHI recognizes the sequence GGATCC and cuts the DNA as indicated.

DNA can be cut by restriction enzymes in two different ways.

One way is to cut DNA to leave overhangs of unpaired DNA based, or “sticky” ends. They are called sticky ends because the DNA bases of each unpaired end have an affinity for each other based on complementary base-pairing rules. This is shown below.

Another way is to cut DNA to leave a “blunt” end in which there are no overhangs, as seen below.

Since the example question is showing the cut DNA leaving overhangs, this is a sticky-end cut. Sticky ends can be useful because similarly cut DNA sequences can be combined to form new DNA molecules.

Therefore, the correct answer is option C, sticky ends.

As far as biotechnology applications are concerned, this “sticky ends” feature of restriction enzymes can be used to our advantage. Recombinant DNA can be created by “cutting and pasting” DNA from multiple organisms that share the same sticky ends. The applications for this are endless!

For example, suppose we were studying a new protein in a certain species of fish and wanted to know what part of the fish’s body the protein localized to. With restriction enzymes, we could create recombinant DNA to glue together the gene for a fluorescent signal protein with the gene for a fish protein. Then, we can sit back and watch where the fish protein is produced! This is outlined in Figure 6.

In this example we can see below that this protein is localized in the brain of the fish, meaning this protein might have some impact on brain functions.

Fluorescent fish

Figure7

There are two ways that restriction enzymes can cut DNA. Not all restriction enzymes can produce sticky ends: some will cut their recognition sequence in such a way that there are no overhangs or unpaired DNA sequences. These are called blunt ends. These can still be used to produce recombinant DNA, but they are not as efficient as using restriction enzymes that make sticky ends.

Key Term: Blunt Ends

Restriction enzymes can cut DNA to produce “blunt ends,” which have no unpaired DNA sequences, and both terminate with a base pair. For example, 5335.-GATCTGACTGATGC--CTAGACTGACTACG-

Example 5: Recognizing Blunt-End Cuts

The mechanism of Hpal is demonstrated in the diagram provided. What term is given to the cuts that Hpal leaves?

  1. Open ends
  2. Blunt ends
  3. Sticky ends
  4. Closed ends
  5. Short ends

Answer

Restriction enzymes are like molecular scissors that cut DNA into fragments. They are able to recognize specific sequences of DNA that are called recognition sequences. Each restriction enzyme has its own unique recognition sequence. In this example, the restriction enzyme HpaI recognizes the sequence GTTAAC and cuts the DNA as indicated.

DNA can be cut by restriction enzymes in two different ways.

One way is for the DNA to be cut to leave a “blunt” end in which there are no overhangs, as seen below.

The other way is to cut DNA to leave overhangs of unpaired DNA bases, or “sticky,” ends. They are called sticky ends because the DNA bases of each unpaired end have an affinity for each other based on complementary base-pairing rules. This is shown below.

Since the example question is showing the cut DNA leaving no overhangs, this is a blunt end cut. Therefore, the correct answer is option B, blunt ends.

Let’s recap some of the key points we have covered in this explainer.

Key Points

  • Restriction enzymes are naturally found in bacteria to defend against viral infections.
  • They bind to specific DNA sequences, called recognition sequences, and can cut DNA.
  • Recognition sequences can be palindromes, meaning they read the same in the 5 to 3 direction on both DNA strands.
  • Restriction enzymes can cut DNA to produce either sticky ends or blunt ends.
  • Sticky ends can be useful in creating recombinant DNA.

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