6.3 Restriction Endonucleases

Restriction endonucleases cleave at specific sequences of double-stranded DNAs. These enzymes were discovered in various bacteria in the early seventies. At present, more than 800 restriction endonucleases have been isolated from different (hundreds of) bacterial strains. All restriction endonucleases belong to three major classes (I, II and III). Of particular interest to gene engineers are restriction endonucleases of type II, recognizing 4- to 8-nucleotide sequences. In such segments, they cleave the double-stranded DNA chain at linkages located either in the middle or at the edges of the recognition site. Usually, but not always, the sequences recognized by restriction endonucleases are symmetrical (have a second-order axis of symmetry). The cleavage of each chain proceeds at identical internucleotide linkages with respect to the axis of symmetry. Shown schematically below are nucleotide sequences (commonly called sites) recognized by some restriction endonucleases. The arrows indicate the internucleotide linkages that undergo cleavage.

It can be seen that certain enzymes cleave DNA in such a way that the duplex portions at the cleavage sites have protruding 5' (EcoRI) or 3' (HhaI, PstI) ends. In other instances (HaeIII), products with blunt ends are formed. The recognized tetranucleotide sequences occur at intervals equal to 4⁴ = 256 nucleotides, on the average, while the hexanucleotide ones occur at intervals equal to 4⁶ = 4096 nucleotides. Consequently, the cleavage of DNA by a restriction endonuclease recognizing hexanucleotide sites produces longer fragments. Significantly, since restriction endonucleases recognize strictly defined portions of DNA, they thus provide a highly precise tool for controlled cleavage of DNA into fragments of predetermined lengths having, in turn, strictly defined termini. Summarized in Table 6-2 are some restriction endonucleases used in structural studies.

Table 6-2. Some Restriction Enzymes Used for Analyzing the Primary DNA Structure. The enzymes recognizing the same nucleotide sequences are called isoschizomers. The arrows indicate the internucleotide linkages that undergo cleavage. In the case of doublestranded oligodeoxyribonucleotide sequences, the polarity of the ends is always:
5'-GGCC-3'
3'-CCGG-5'
The same applies to other sequences as well.