How Do Restriction Enzymes Cut DNA Sequences?

restriction enzymes
Restriction enzymes are enzymes that cut DNA into fragments based upon recognizing a specific sequence of nucleotides. Restriction enzymes are also known as restriction endonucleases.

 Callista Images/Cultura/Getty Images

In nature, organisms constantly have to protect themselves from foreign invaders, even at the microscopic level. In bacteria, there is a group of bacterial enzymes that work by dismantling foreign DNA. This dismantling process is called restriction and the enzymes that carry out this process are called restriction enzymes.

Restriction enzymes are very important in recombinant DNA technology. Restriction enzymes have been used to help produce vaccines, pharmaceutical products, insect resistant crops, and a host of other products.

Key Takeaways

  • Restriction enzymes dismantle foreign DNA by cutting it into fragments. This disassembling process is called restriction.
  • Recombinant DNA technology relies on restriction enzymes to produce new combinations of genes.
  • The cell protects its own DNA from disassembly by adding methyl groups in a process called modification.
  • DNA ligase is a very important enzyme that helps to join DNA strands together via covalent bonds.

What Is a Restriction Enzyme?

Restriction enzymes are a class of enzymes that cut DNA into fragments based upon recognizing a specific sequence of nucleotides. Restriction enzymes are also known as restriction endonucleases.

While there are hundreds of different restriction enzymes, they all work in essentially the same way. Each enzyme has what is known as a recognition sequence or site. A recognition sequence is typically a specific, short nucleotide sequence in DNA. The enzymes cut at certain points within the recognized sequence. For example, a restriction enzyme may recognize a specific sequence of guanine, adenine, adenine, thymine, thymine, cytosine. When this sequence is present, the enzyme can make staggered cuts in the sugar-phosphate backbone in the sequence.

But if restriction enzymes cut based on a certain sequence, how do cells like bacteria protect their own DNA from being cut up by restriction enzymes? In a typical cell, methyl groups (CH3) are added to the bases in the sequence to prevent recognition by the restriction enzymes. This process is carried out by complementary enzymes that recognize the same sequence of nucleotide bases as restriction enzymes. The methylation of DNA is known as modification. With the processes of modification and restriction, cells can both cut up foreign DNA that pose a danger to the cell while preserving the important DNA of the cell.

Based on the double-stranded configuration of DNA, recognition sequences are symmetrical on the different stands but run in opposite directions. Recall that DNA has "direction" indicated by the type of carbon at the end of the strand. The 5' end has a phosphate group attached while the other 3' end has a hydroxyl group attached. For example:

5' end - ... guanine, adenine, adenine, thymine, thymine, cytosine ... - 3' end

3' end - ... cytosine, thymine, thymine, adenine, adenine, guanine ... - 5' end

If, for example, the restriction enzyme cuts within the sequence between the guanine and adenine, it would do so with both sequences but at opposite ends (since the second sequence runs in the opposite direction). Since the DNA is cut on both strands, there will be complementary ends that can hydrogen bond to one another. These ends are often called "sticky ends."

What Is DNA Ligase?

The sticky ends of the fragments produced by restriction enzymes are useful in a laboratory setting. They can be used to join DNA fragments from both different sources and different organisms. The fragments are held together by hydrogen bonds. From a chemical perspective, hydrogen bonds are weak attractions and are not permanent. Using another type of enzyme however, the bonds can be made permanent.

DNA ligase is a very important enzyme that functions in both the replication and repair of a cell's DNA. It functions by helping the joining of DNA strands together. It works by catalyzing a phosphodiester bond. This bond is a covalent bond, much stronger than the aforementioned hydrogen bond and able to hold the different fragments together. When different sources are used, the resulting recombinant DNA that is produced has a new combination of genes.

Restriction Enzyme Types

There are four broad categories of restriction enzymes: Type I enzymes, Type II enzymes, Type III enzymes, and Type IV enzymes. All have the same basic function, but the different types are classified based on their recognition sequence, how they cleave, their composition, and on their substance requirements (the need for and type of cofactors). Generally, Type I enzymes cut DNA at locations distant to the recognition sequence; Type II cut DNA within or close to the recognition sequence; Type III cut DNA near recognition sequences; and Type IV cleave methylated DNA.

Sources

  • Biolabs, New England. “Types of Restriction Endonucleases.” New England Biolabs: Reagents for the Life Sciences Industry, www.neb.com/products/restriction-endonucleases/restriction-endonucleases/types-of-restriction-endonucleases.
  • Reece, Jane B., and Neil A. Campbell. Campbell Biology. Benjamin Cummings, 2011.