Restriction enzymes played a critical role in the advent of genetic engineering. In this lesson, you will learn what role restriction enzymes play in creating recombinant DNA.
Restriction enzymes may have evolved to fight invading viruses.
The goal of genetic engineering is changing the genetic makeup of an organism. To achieve this goal, scientists must have a way of rearranging genes to create new combinations of DNA. Restriction enzymes are one tool that can be used to accomplish this goal.
What Is a Restriction Enzyme?
So, what is a restriction enzyme? A restriction enzyme is an enzyme that cuts DNA after recognizing a specific sequence of DNA. You can think of restriction enzymes as molecular scissors. Scientists can use restriction enzymes to cut a single gene from a larger piece of DNA. Restriction enzymes evolved in bacteria.
Now you may be thinking to yourself, 'That seems like something that's too good to be true.' Well, nature didn't create this enzyme just so humans would have a laboratory tool to manipulate DNA. Bacteria, like us, fight a constant battle against viruses. Scientists believe restriction enzymes evolved to protect bacteria from invading viruses.
By recognizing a sequence in viral DNA and cutting the DNA molecule, restriction enzymes inhibit, or restrict, viral infections of bacteria. Let's see how restriction enzymes accomplish that goal.
The restriction enzyme identifies the recognition sequence and cuts it out.
One of the key traits of a restriction enzyme that makes it so valuable to scientists is the fact that restriction enzymes cut at or near a specific sequence of DNA. The DNA sequence recognized by a restriction enzyme is known as the recognition sequence. Let's look at a specific example to see how this works.
The restriction enzyme EcoRI recognizes the sequence GAATTC. Whenever EcoRI sees this particular six-nucleotide sequence, it's going to make a cut in the DNA. Recall that DNA is always read from the 5' end to the 3' end of the strand. If you look more closely at the recognition site, you should notice that the recognition sequence is the same in both strands. If you read the top strand from the 5' to the 3' end, the strand reads GAATTC. However, if you read the bottom strand from the 5' end to the 3' end, you get the same result: GAATTC.
When a nucleotide sequence is the same whether it is read forward or backward, it's called a palindromic sequence.
You may be more familiar with palindromes in a composition class, where it is defined as a word or sentence that reads the same in both directions. A classic example of a palindromic sentence is, 'A man, a plan, a canal, Panama.'
Let's briefly consider this example. If you start with the last letter of the sentence and work your way toward the first letter, you end up with the same sentence: A man, a plan, a canal, Panama. Note that like the DNA sequence, reading the sentence forward or backward yields the same result.
A palindromic sequence reads the same forward and backward.
How Does It Work?
Great. So, now we know how a restriction enzyme determines where to cut DNA. But, exactly what is it cutting? Recall that there are two types of bonds in a DNA molecule that it could be breaking: covalent bonds and hydrogen bonds. Hydrogen bonds hold complementary nucleotide bases together. Covalent bonds between the sugar and phosphate groups hold the DNA backbone together.
If we think of the DNA strand as a paper streamer to be cut by scissors, should a restriction enzyme cut the hydrogen bonds or covalent bonds to achieve this goal? 'Cutting' the hydrogen bonds would be the equivalent of cutting the streamer straight down the middle. That will not chop our streamer into shorter pieces. Cutting the streamer in that orientation will simply create two skinnier streamers of the same length.
On the other hand, cutting covalent bonds would be the equivalent of chopping the streamer into shorter pieces. Most restriction enzymes cut a specific covalent bond within the recognition sequence. Our example enzyme, EcoRI, breaks the covalent bond between the guanine and first adenine nucleotide in the recognition sequence.
Note the effect the palindromic recognition sequence has on the DNA fragments that are produced by a restriction enzyme. The DNA molecule has not been cut cleanly in half at a single location. The breaks made in the DNA backbone are offset. That means that cutting the DNA molecule has produced single-stranded DNA sequences at the ends of the resulting strands. These single-stranded overhangs are known as sticky ends.
You might think that is a peculiar name for a piece of DNA. The reason it is called a sticky end is that single-stranded DNA is less stable than double-stranded DNA. That means that single-stranded nucleotides will try to form hydrogen bonds with complementary nucleotides to create a more stable molecule.
Single-stranded DNA sequence overhangs are called sticky ends.
Recall that G binds with C and A binds with T. This is another important characteristic restriction enzymes provide scientists: specificity. Because nucleotide bases will only hydrogen-bond with their complement, sticky ends only bond with a complementary sticky end. For instance, in our EcoRI example, the TTAA will be able to hydrogen-bond with the TTAA of the complementary strand.
It is important to note that we have explored one particular example of a restriction enzyme. Not all restriction enzymes create sticky ends when cutting DNA, nor do they always cut within the recognition sequence. This particular restriction enzyme was chosen to highlight the role restriction enzymes can play in creating recombinant DNA, such as the insulin plasmid in our example.
We will soon see how all of these characteristics of restriction enzymes play an important role in genetic engineering experiments.
In summary, a restriction enzyme is an enzyme that cuts DNA after recognizing a specific sequence of DNA. You can think of a restriction enzyme as molecular scissors that cleave the covalent bond linking adjacent nucleotides in the backbone of a DNA strand.
A recognition sequence is the DNA sequence recognized by a restriction enzyme. The recognition sites of most enzymes used by scientists are palindromic. A palindromic sequence is a nucleotide sequence that is the same when read 5' to 3' in either strand. A sticky end is a single-stranded DNA overhang at the end of a DNA molecule.
At the conclusion of this video, you should be able to:
- Define restriction enzyme, recognition sequence, palindromic sequence and sticky end
- Explain the function of restriction enzymes