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.
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