What are restriction enzymes? |

Discovery and Role of Restriction Enzymes in Bacteria

Nucleases are a broad class of enzymes that destroy nucleic acids by breaking the sugar-phosphate backbone of the molecule. Until 1970, the only known nucleases were those that destroyed nucleic acids nonspecifically—that is, in a random fashion. For this reason, these enzymes were of limited usefulness for working with nucleic acids such as DNA and RNA. In 1970, molecular biologist Hamilton O. Smith discovered a type of nuclease that could fragment DNA molecules in a specific and therefore predictable pattern. This nuclease, HindII, was the first restriction endonuclease, or restriction enzyme. Smith was working with the bacterium Haemophilus influenzae (H. influenzae) when he discovered this enzyme, which is capable of destroying DNA from other bacterial species but not the DNA of H. influenzae itself. The term “restriction” refers to the apparent role these enzymes play in destroying the DNA of invading bacteriophages
(bacterial viruses) while leaving the bacterial cell’s own DNA untouched. A bacterium with such an enzyme was said to “restrict” the host range of the bacteriophage.

As more restriction enzymes from a wide variety of bacterial species were discovered in the 1970s, it became increasingly clear that these enzymes could be useful for creating and manipulating DNA fragments in unique ways. What was not clear was how these enzymes were able to distinguish between bacteriophage DNA and the bacterial cell’s own DNA. A chemical comparison between DNA that could and could not be fragmented revealed that the DNA molecules differed slightly at the restriction sites (the locations the enzyme recognized and cut). Nucleotides at the restriction site were found to have methyl functional groups (–CH3) attached to them, giving this phenomenon the name DNA methylation.

The conclusion was that the methylation somehow protected the DNA from attack, which could account for Smith’s observation that H. influenzae DNA was not destroyed by its own restriction enzyme; presumably the enzyme recognized a specific methylation pattern on the DNA molecule and left it alone. Foreign DNA, such as that from another species, would not have the correct methylation pattern, or might not be methylated at all, and could therefore be fragmented by the restriction enzyme. Hence, restriction enzymes are now regarded as part of a simple yet effective bacterial defense mechanism to guard against foreign DNA, which can enter bacterial cells with relative ease.

Mechanism of Action

To begin the process of cleaving a DNA molecule, a restriction enzyme must first recognize the appropriate place on the molecule. The recognition site for most restriction enzymes involves a short, usually four- to six-nucleotide palindromic sequence. A palindrome is a word or phrase that reads the same backward and forward, such as “Otto” or “madam”; in terms of DNA, a palindromic sequence is one that reads the same on each strand of DNA but in opposite directions. EcoRI, derived from the bacterium Escherichia coli, is an example of an enzyme that has a recognition site composed of nucleotides arranged in a palindromic sequence:
——GAATTC————CTTAAG——

Whether the top sequence is read from left to right or the bottom sequence is read from right to left, it is always GAATTC.

An additional consideration in the mechanism of restriction-enzyme activity is the type of cut that is made. When a restriction enzyme cuts DNA, it is actually breaking the “backbone” of the molecule, which consists of a chain of sugar and phosphate molecules. This breakage occurs at a precise spot on each strand of the double-stranded DNA molecule. The newly created ends of the DNA fragments are informally referred to as either “sticky ends” or “blunt ends,” depending on whether or not single-stranded regions of DNA are generated by the cutting activity of the restriction enzyme. For example, the enzyme EcoRI is a sticky-end cutter; when the cuts are made at the recognition site, the result is:
—GAATTC—  —G  AATTC—
→  
—CTTAAG—  —CTTAA  G—

The break in the DNA backbone is made just after the G in each strand; this helps weaken the connections between the nucleotides in the middle of the site, and the DNA molecule splits into two fragments. The single-stranded regions, where the bases TTAA are not paired with their complements (AATT) on the other strand, are called overhangs; however, the bases in one overhang are still capable of pairing with the bases in the other overhang, as they did before the DNA strands were cut. The ends of these fragments will readily stick to each other if brought close together—hence the name “sticky ends.”

Enzymes that create blunt ends make a flush cut and do not leave any overhangs, as demonstrated by the cutting site of the enzyme AluI:
——AGCT——  ——AG  CT——
→  
——TCGA——  ——TC  GA——

Because of the lack of overhanging single-strand regions, these two DNA fragments will not readily rejoin. In practice, either type of restriction enzyme may be used, but enzymes that produce sticky ends are generally favored over blunt-end cutters because of the ease with which the resulting fragments can be rejoined.

Impact and Applications

It is no exaggeration to say that the entire field of genetic engineering would have been impossible without the discovery and widespread use of restriction enzymes. On the most basic level, restriction enzymes allow scientists to create recombinant DNA
molecules (hybrid molecules containing DNA from different sources, such as humans and bacteria). No matter what the source, DNA molecules can be cut with restriction enzymes to produce fragments that can then be rejoined in new combinations with DNA fragments from other molecules. This technology has led to advances such as the production of human insulin by bacterial cells such as Escherichia coli.

The DNA of most organisms is relatively large and complex; in fact, it is usually so large that it becomes difficult to manipulate and study the DNA of some organisms, such as humans. Restriction enzymes provide a convenient way to cut large DNA molecules very specifically into smaller fragments that can then be used more easily in a variety of molecular genetics procedures.

Another area of genetic engineering that is possible because of restriction enzymes is the production of restriction maps. A restriction map is a diagram of a DNA molecule showing where particular restriction enzymes cut the molecule and the molecular sizes of the fragments that are generated. The restriction sites can then be used as markers for further study of the DNA molecule and to help geneticists locate important genetic regions. Use of restriction enzymes has also revealed other interesting and useful markers of the human genome, called restriction fragment length polymorphisms (RFLP). The name refers to changes in the size of restriction fragments caused by mutations in the recognition site for a particular restriction enzyme. The recognition site is mutated so that the restriction enzyme no longer cuts there, resulting in one long fragment where, before the mutation, there would have been two shorter fragments. These changes in fragment length can then be used as markers for the region of DNA in question. Because they result from mutations in the DNA sequence, they are inherited from one generation to the next. Thus, these mutations have been a valuable tool for molecular biologists mapping human DNA and for those scientists involved in “fingerprinting” individuals by means of their DNA.

Key Terms




enzyme


:

a molecule, usually a protein, that is used by cells to facilitate and speed up a chemical reaction




methylation

:

the process of adding a methyl functional group (one carbon atom and three hydrogen atoms) to a particular molecule, such as a DNA nucleotide




nuclease

:

a type of enzyme that breaks down the sugar-phosphate backbone of nucleic acids such as DNA and RNA





nucleotides


:

the building blocks of nucleic acids, composed of a sugar, a phosphate group, and a nitrogen-containing base





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