Restriction Endonucleases

RESTRICTION ENDONUCLEASES

One of the major obstacles to molecular analysis of genomic DNA is the immense size of the molecules involved. The discovery of a special group of bacterial enzymes, called restriction endonucleases (restriction enzymes), which cleave double-stranded (ds) DNA into smaller, more manageable fragments, opened the way for DNA analysis. Because each enzyme cleaves DNA at a specific nucleotide sequence (restriction site), restriction enzymes are used experimentally to obtain precisely defined DNA segments called restriction fragments.

A. Specificity of restriction endonucleases

Restriction endonucleases recognize short stretches of dsDNA (four to eight bp) that contain specific nucleotide sequences. These sequences, which differ for each restriction enzyme, are palindromes, that is, they exhibit twofold rotational symmetry (Figure 33.2). This means that, within a short region of the double helix, the nucleotide sequence on the two strands is identical if each is read in the 5→3I direction. Therefore, if you turn the page upside down (that is, rotate it 180° around its axis of symmetry) the sequence remains the same.

In bacteria, restriction endonucleases “restrict” the expression of nonbacterial (foreign) DNA through cleavage. Bacterial DNA is protected by methylation of bases at the restriction site.

Figure 33.2 Recognition sequence of restriction endonuclease EcoRI shows twofold rotational symmetry. dsDNA = double-stranded DNA; A = adenine; C = cytosine; G = guanine; T = thymine.

B. Nomenclature

A restriction enzyme is named according to the organism from which it was isolated. The first letter of the name is from the genus of the bacterium. The next two letters are from the name of the species. An additional letter indicates the type or strain, and a number (Roman numeral) is appended to indicate the order in which the enzyme was discovered in that particular organism. For example, HaeIII is the third restriction endonuclease isolated from the bacterium Haemophilus aegyptius.

C. “Sticky” and “blunt” ends

Restriction enzymes cleave dsDNA so as to produce a 3I -hydroxyl group on one end and a 5I -phosphate group on the other. Some restriction endonucleases, such as TaqI, form staggered cuts that produce “sticky” or cohesive ends (that is, the resulting DNA fragments have single-stranded sequences that are complementary to each other) as shown in Figure 33.3. Other restriction endonucleases, such as HaeIII, produce fragments that have “blunt” ends that are double stranded and, therefore, do not form hydrogen bonds with each other. Using the enzyme DNA ligase, sticky ends of a DNA fragment of interest can be covalently joined with other DNA fragments that have sticky ends produced by cleavage with the same restriction endonuclease (Figure 33.4). [Note: A ligase encoded by bacteriophage T4 can covalently join blunt-ended fragments.]

Figure 33.3 Specificity of TaqI and HaeIII restriction endonucleases; A = adenine; C = cytosine; G = guanine; T = thymine.

Figure 33.4 Formation of recombinant DNA from restriction fragments with “sticky” ends. A = adenine; C = cytosine; G = guanine; T = thymine.

D. Restriction sites

A DNA sequence that is recognized and cut by a restriction enzyme is called a restriction site. Restriction endonucleases cleave dsDNA into fragments of different sizes depending upon the size of the sequence recognized. For example, an enzyme that recognizes a specific 4-bp sequence produces many cuts in the DNA molecule, one every 4⁴ bp. In contrast, an enzyme requiring a unique sequence of 6 bp produces fewer cuts (one every 4⁶ bp) and, therefore, longer pieces. Hundreds of these enzymes, each having different cleavage specificities (varying in both nucleotide sequences and length of recognition sites), are commercially available.