Discovery and Role of Restriction Enzymes in Bacteria (Genetics & Inherited Conditions)
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 was 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 1970’s, it became increasingly clear that these enzymes could be useful for creating and manipulating DNA fragments in unique ways. What was not clear, however, was...
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Mechanism of Action (Genetics & Inherited Conditions)
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——
If 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, consisting 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 then informally referred to as “sticky ends” or “blunt ends.” These terms refer to whether single-stranded regions of DNA are generated by the cutting activity of the restriction enzyme. For example, the...
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Impact and Applications (Genetics & Inherited Conditions)
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; it is usually so large, in fact, 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 fragments that are generated. The restriction sites can then be used as markers for further study of the DNA molecule and to...
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Further Reading (Genetics & Inherited Conditions)
Allison, Lizabeth A. “Recombinant DNA Technology and Molecular Cloning.” In Fundamental Molecular Biology. Malden, Mass.: Blackwell, 2007. Includes information about cutting and joining DNA and RFLP.
Drlica, Karl. Understanding DNA and Gene Cloning: A Guide for the Curious. 4th ed. New York: John Wiley and Sons, 2004. Provides basic information about restriction enzymes and their use in cloning. Illustrations, bibliography, index.
Karp, Gerald. “Techniques in Cell Molecular Biology.” In Cell and Molecular Biology: Concepts and Experiments. 5th ed. Chichester, England: John Wiley and Sons, 2008. Includes information about restricted enzymes.
Lewin, Benjamin. Genes IX. Sudbury, Mass.: Jones and Bartlett, 2007. Provides a detailed yet highly readable explanation of restriction and methylation in bacteria. Illustrations, bibliography, index.
Watson, James D., et al. Recombinant DNA—Genes and Genomes: A Short Course. 3d ed. New York: W. H. Freeman, 2007. An excellent resource for the general reader wishing to understand the basics of genetic engineering. Illustrations, diagrams, bibliography, index.
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Web Sites of Interest (Genetics & Inherited Conditions)
Access Excellence, Biotech Chronicles. http://www.accessexcellence.org/AE/AEC/CC/restriction.php. Access Excellence, a national science education program, includes a background paper on restriction enzymes, as well as a chart listing examples of restriction enzymes, a classroom activity, and other information on restriction enzymes.
Dolan DNA Learning Center, DNA Restriction. http://www.dnalc.org/resources/animations/restriction.html. The site’s biology animation library offers a brief definition of DNA restriction and enables users to download an animation that demonstrates the process.
Kimball’s Biology Pages. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RestrictionEnzymes.html. John Kimball, a retired Harvard University biology professor, includes a page about restriction enzymes, with links to information about recombinant DNA, in his online cell biology text.
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Restriction Enzymes (World of Microbiology and Immunology)
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'AATTC', 3'TTAAG'. 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
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