Restriction Enzymes

Restriction enzymes are bacterial proteins that recognize specific DNA sequences and cut DNA at or near the recognition site. These enzymes are widely used in molecular genetics for analyzing DNA and creating recombinant DNA molecules.

Biological Function and Historical Background

Restriction enzymes apparently evolved as a primitive immune system in bacteria. If viruses enter a bacterial cell containing restriction enzymes, the viral DNA is fragmented. Destruction of the viral DNA prevents destruction of the bacterial cell by the virus. The term "restriction" derives from the phenomenon in which bacterial viruses are restricted from replicating in certain strains of bacteria by enzymes that cleave the viral DNA, but leave the bacterial DNA untouched. In bacteria, restriction enzymes form a system with modification enzymes that methylate the bacterial DNA. Methylation of DNA at the recognition sequence typically protects the microbe from cleaving its own DNA.

Since the 1970s, restriction enzymes have had a very important role in recombinant DNA techniques, in both the creation and analysis of recombinant DNA molecules. The first restriction enzyme was isolated and characterized in 1968, and over 3,400 restriction enzymes have been discovered since. Of these enzymes, over 540 are currently commercially available.

Nomenclature and Classification

Restriction enzymes are named based on the organism in which they were discovered. For example, the enzyme Hin d III was isolated from Haemophilus influenzae, strain Rd. The first three letters of the name are italicized because they abbreviate the genus and species names of the organism. The fourth letter typically comes from the bacterial strain designation. The Roman numerals are used to identify specific enzymes from bacteria that contain multiple restriction enzymes. Typically, the Roman numeral indicates the order in which restriction enzymes were discovered in a particular strain.

There are three classes of restriction enzymes, labeled types I, II, and III. Type I restriction systems consist of a single enzyme that performs both modification (methylation) and restriction activities. These enzymes recognize specific DNA sequences, but cleave the DNA strand randomly, at least 1,000 base pairs (bp) away from the recognition site. Type III restriction systems have separate enzymes for restriction and methylation, but these enzymes share a common subunit. These enzymes recognize specific DNA sequences, but cleave DNA at random sequences approximately twenty-five bp from the recognition sequence. Neither type I nor type III restriction systems have found much application in recombinant DNA techniques.

Type II restriction enzymes, in contrast, are heavily used in recombinant DNA techniques. Type II enzymes consist of single, separate proteins for restriction and modification. One enzyme recognizes and cuts DNA, the other enzyme recognizes and methylates the DNA. Type II restriction enzymes cleave the DNA sequence at the same site at which they recognize it. The only exception are type IIs (shifted) restriction enzymes, which cleave DNA on one side of the recognition sequence, within twenty nucleotides of the recognition site. Type II restriction enzymes discovered to date collectively recognize over 200 different DNA sequences.

Type II restriction enzymes can cleave DNA in one of three possible ways. In one case, these enzymes cleave both DNA strands in the middle of a recognition sequence, generating blunt ends. For example: (The notations 5′ and 3′ are used to indicate the orientation of a DNA molecule. The numbers 5 and 3 refer to specific carbon atoms in the deoxyribose sugar in DNA.)

These blunt ended fragments can be joined to any other DNA fragment with blunt ends, making these enzymes useful for certain types of DNA cloning experiments.

Type II restriction enzymes can also cleave DNA to leave a 3′ ("three prime") overhang. (An overhang means that the restriction enzyme leaves a short single-stranded "tail" of DNA at the site where the DNA was cut.) These 3′ overhanging ends can only join to another compatible 3′ overhanging end (that is, an end with the same sequence in the overhang). Finally, some type II enzymes can generate 5′ overhanging DNA ends, which can only be joined to a compatible 5′ end.

In the type II restriction enzymes discovered to date, the recognition sequences range from 4 bp to 9 bp long. Cleavage will not occur unless the full length of the recognition sequence is encountered. Enzymes with a short recognition sequence cut DNA frequently; restriction enzymes with 8 or 9 bp sequences typically cut DNA very infrequently, because these longer sequences are less common in the target DNA.

Use of Restriction Enzymes in Biotechnology

The ability of restriction enzymes to reproducibly cut DNA at specific sequences has led to the widespread use of these tools in many molecular genetics techniques. Restriction enzymes can be used to map DNA fragments or genomes. Mapping means determining the order of the restriction enzyme sites in the genome. These maps form a foundation for much other genetic analysis. Restriction enzymes are also frequently used to verify the identity of a specific DNA fragment, based on the known restriction enzyme sites that it contains.

Perhaps the most important use of restriction enzymes has been in the generation of recombinant DNA molecules, which are DNAs that consist of genes or DNA fragments from two different organisms. Typically, bacterial DNA in the form of a plasmid (a small, circular DNA molecule) is joined to another piece of DNA (a gene) from another organism of interest. Restriction enzymes are used at several points in this process. They are used to digest the DNA from the experimental organism, in order to prepare the DNA for cloning. Then a bacterial plasmid or bacterial virus is digested with an enzyme that yields compatible ends. These compatible ends could be blunt (no overhang), or have complementary overhanging sequences. DNA from the experimental organism is mixed with DNA from the plasmid or virus, and the DNAs are joined with an enzyme called DNA ligase . As noted above, the identity of the recombinant DNA molecule is often verified by restriction enzyme digestion.

Restriction enzymes also have applications in several methods for identifying individuals or strains of a particular species. Pulsed field gel electrophoresis is a technique for separating large DNA fragments, typically fragments resulting from digesting a bacterial genome with a rare-cutting restriction enzyme. The reproducible pattern of DNA bands that is produced can be used to distinguish different strains of bacteria, and help pinpoint if a particular strain was the cause of a widespread disease outbreak, for example.

Restriction fragment length polymorphism (RFLP) analysis has been widely used for identification of individuals (humans and other species). In this technique, genomic DNA is isolated, digested with a restriction enzyme, separated by size in an agarose gel, then transferred to a membrane. The digested DNA on the membrane is allowed to bind to a radioactively or fluorescently labeled probe that targets specific sequences that are bracketed by restriction enzyme sites. The size of these fragments varies in different individuals, generating a "biological bar code" of restriction enzyme-digested DNA fragments, a pattern that is unique to each individual.

Restriction enzymes are likely to remain an important tool in modern genetics. The reproducibility of restriction enzyme digestion has made these enzymes critical components of many important recombinant DNA techniques.

see also Biotechnology; Cloning Genes; Gel Electrophoresis; Mapping; Methylation; Nucleases; Polymorphisms; Recombinant DNA.

Patrick G. Guilfoile

Bibliography

Bloom, Mark V., Greg A. Freyer, and David A. Micklos. Laboratory DNA Science: An Introduction to Recombinant DNA Techniques and Methods of Genome Analysis. Menlo Park, CA: Addison-Wesley, 1996.

Cooper, Geoffrey. The Cell: A Molecular Approach. Washington, DC: ASM Press, 1997.

Kreuzer, Helen, and Adrianne Massey. Recombinant DNA and Biotechnology, 2nd ed. Washington, DC: ASM Press, 2000.

Lodish, Harvey, et al. Molecular Cell Biology, 4th ed. New York: W. H. Freeman, 2000.

Old, R. W., and S. B. Primrose. Principles of Gene Manipulation, 5th ed. London: Blackwell Scientific Publications, 1994.

Internet Resource

Roberts, Richard J., and Dana Macelis. Rebase. <http://rebase.neb.com>.

Restriction Enzymes

Restriction enzymes (also known as restriction endonucleases) are enzymes that cut double-stranded DNA at very specific recognition sites. They were originally discovered in bacteria that use them to restrict the growth of viruses but are now among the workhorse enzymes of biotechnology and recombinant DNA research.

It has long been known that bacteria are susceptible to attack by viruses known as bacteriophages ("eaters of bacteria"). The presence of restriction enzymes in bacteria is part of the defense system against bacteriophages that has evolved in these bacteria. These highly specific enzymes will scan DNA until a certain sequence of nucleotide bases is identified. The specificity is such that the sequence is apt to occur at only one or two sites in the viral DNA, with no such occurrence in the host bacterial DNA. Restriction enzymes recognize a sequence such as:

5′ G A A T T C 3′

3′ C T T A A G 5′

Interestingly, the enzyme can recognize the paired sequences from either strand because they are a palindrome (reading the same in either direction). The restriction enzyme can cut this palindromic sequence in one of two manners: across both strands at the same spot, or in a staggered manner that yields free single-stranded ends called "sticky ends." These sticky ends have proved most useful in recombinant DNA work.

The names of restriction enzymes are derived from their bacterial sources. One of the enzymes most widely used in recombinant DNA work is Eco R1, which is isolated from Escherichia coli RY13. Other examples include HindII (isolated from Haemophilus influenza Rd), and Xba I (isolated from Xanthomonas badrii ). The specificity of each enzyme allows researchers to cut DNA in a predictable and reproducible manner. Using Eco R1 on the above sequence, one would always obtain the ends:

————– 5′ G     A A T T C 3′ ————–

————– 3′ C T T A A      G 5′ ————–

A small circular piece of DNA (such as a bacterial plasmid) with one Eco R1 site would yield a linear piece of DNA with the CTTAA sticky ends. Now suppose one could obtain the DNA (the gene) coding for a foreign protein such as human insulin. At each end of the insulin gene, one could attach complementary single-stranded sticky ends that would exactly "match" the ends of the cut plasmid. As if made of Velcro, the complementary ends would stick together, and if one were to use an enzyme called DNA ligase to form a stronger covalent bond between parts joined together, one would have incorporated a human (or other species) gene into bacterial DNA. This technique is known as recombinant DNA. The bacteria can now be grown in large batches and made to synthesize the foreign protein (insulin).

see also Deoxyribonucleic Acid; Enzyme; Recombinant Dna.

C. Larry Bering

Bibliography

Voet, Donald, and Voet, Judith G. (1995). Biochemistry, 2nd edition New York: John Wiley.

Internet Resources

Peters, Pamela. "Restriction Enzymes Background Paper." Access Excellence Activities Exchange. Available from <http://www.accessexcellence.org>.

Restriction enzymes

Restriction enzymes are proteins that are produced by bacteria as a defense mechanism against viruses that infect the bacteria (bacterial phages). Most bacteria have restriction modification systems that consist of methylases and restriction enzymes. In such systems a bacteria's own DNA is modified by methylation (the addition of a methyl group, CH ₃) at a specific location determined by a specific pattern of nucleotide residue and protected from degradation by specialized enzymes termed endonucleases.

The names of restriction enzymes are created from the first letter of the bacterial genus followed by the first two letters of the species plus a Roman numeral if more than one restriction enzyme has been identified in a particular species. Thus, the fifth restriction enzyme from E. coli is called EcoRV (pronounced e, ko, r five). Besides cloning , restriction enzymes are used in genetic mapping techniques, linking the genome directly to a conventional genetic marker.

Any DNA molecule, from viruses to humans, contains restriction-enzyme target sites purely by chance and, therefore, may be cut into defined fragments of size suitable for cloning. Restriction sites are not relevant to the function of the organism, nor would they be cut in vivo, because most organisms do not have restriction enzymes.

There are three types of restriction endonucleases in bacteria. Type I cuts unmodified DNA at a non-specific site 1000 base pairs beyond the recognition site. Type III recognizes a short asymmetric sequence and cuts at a site 24-26 base pairs from the recognition site. Type II recognizes short DNA of four to eight nucleotides. Type II restriction enzymes are widely used in molecular biology . Type II restriction enzymes have two properties useful in recombinant DNA technology. First, they cut DNA into fragments of a size suitable for cloning. Second, many restriction enzymes make staggered cuts generating single-stranded ends conducive to the formation of recombinant DNA. Hamilton Smith identified the first type II restriction enzyme, HindII, in 1970 at Johns Hopkins University.

Most type II restriction endonucleases cut DNA into staggered ends. For example, restriction enzyme EcoRI (from the bacterium Escherichia coli ) recognizes the following six-nucleotide-pair sequence in the DNA of any organism: 5'–GAATTC–3', 3'–CTTAAG–5'. This type of segment is called a DNA palindrome, which means that both strands have the same nucleotide sequence but in antiparallel orientation. EcoRI cuts in the six-base-pair DNA between the G and the A nucleotides. This staggered cut leaves a pair of identical single stranded ends. Some enzymes cut DNA at the same position of both strands, leaving both ends blunt.

See also Cell cycle (prokaryotic), genetic regulation of; DNA (Deoxyribonucleic acid); Gene amplification; Gene; Genetic code; Genetic identification of microorganisms; Genetic mapping; Genetic regulation of eukaryotic cells; Molecular biology and molecular genetics