Restriction Endonuclease - Enzymes for cutting DNA

Restriction Enzymes
Restriction endonuclceases Gene cloning requires that DNA molecules be cut in a very precise and reproducible fashion. This is illustrated by the way in which the vector is cut during construction of a recombinant DNA molecule. Each vector molecule must be cleaved at a single position, to open up the circle so that new DNA can be inserted: a molecule that is cut more than once will be broken into two or more separate fragments and will be of no use as a cloning vector. Furthermore, each vector molecule must be cut at exactly the same position on the circle, random cleavage is not satisfactory.
Often it is also necessary to cleave the DNA that is to be cloned. There are two reasons for this. First, if the aim is to clone a single gene, which may consist of only 2 or 3 kb of DNA, then that gene will have to be cut out of the large (often greater than 80 kb) DNA molecules. Second, large DNA molecules may have to be broken down simply to produce fragments small enough to be carried by the vector. Most cloning vectors exhibit a preference for DNA fragments that fall into a particular size range: most plasmid-based vectors, for example, are very inefficient at cloning DNA molecules more than 8 kb in length. Purified restriction endonucleases allow the molecular biologist to cut DNA molecules in the precise, reproducible manner required for gene cloning.
The initial observation that led to the eventual discovery of restriction endonucleases was made in the early 1950s, when it was shown that some strains of bacteria are immune to bacteriophage infection, a phenomenon referred to as host-controlled restriction. The mechanism of restriction is not very complicated, even though it took over 20 years to be fully understood. Restriction occurs because the bacterium produces an enzyme that degrades the phage DNA before it has time to replicate and direct synthesis of new phage particles . The bacterium’s own DNA, the destruction of which would of course be lethal, is protected from attack because it carries additional methyl groups that block the degradative enzyme action. These degradative enzymes are called restriction endonucleases and are synthesized by many, perhaps all, species of bacteria: over 2500 different ones have been isolated and more than 300 are available for use in the laboratory.
Three different classes of restriction endonuclease are recognized, each distinguished by a slightly different mode of action. Types I and III are rather complex and have only a limited role in genetic engineering. Type II restriction endonucleases, on the other hand, are the cutting enzymes that are so important in gene cloning.
The central feature of type II restriction is that each enzyme has a specific recognition sequence at which it cuts a DNA molecule. A particular enzyme cleaves DNA at the recognition sequence and nowhere else. For example, the restriction endonuclease called PvuI (isolated from Proteus vulgaris) cuts DNA only at the hexanucleotide CGATCG. In contrast, a second enzyme from the same bacterium, called PvuII, cuts at a different hexanucleotide, in this case CAGCTG. Many restriction endonucleases recognize hexanucleotide target sites, but others cut at four, five, eight, or even longer nucleotide sequences. Sau3A (from Staphylococcus aureus strain 3A) recognizes GATC, and AluI (Arthrobacter luteus) cuts at AGCT. There are also examples of restriction endonucleases with degenerate recognition sequences, meaning that they cut DNA at any one of a family of related sites. HinfI (Haemophilus influenzae strain Rf), for instance, recognizes GANTC, so cuts at GAATC, GATTC, GAGTC, and GACTC.
The exact nature of the cut produced by a restriction endonuclease is of considerable importance in the design of a gene cloning experiment. Many restriction endonucleases make a simple double-stranded cut in the middle of the recognition sequence , resulting in a blunt end or flush end. PvuII and AluI are examples of blunt end cutters. Other restriction endonucleases cut DNA in a slightly different way. With these enzymes the two DNA strands are not cut at exactly the same position. Instead the cleavage is staggered, usually by two or four nucleotides, so that the resulting DNA fragments have short single-stranded overhangs at each end . These are called sticky or cohesive ends, as base pairing between them can stick the DNA molecule back together again.
One important feature of sticky end enzymes is that restriction endonucleases with different recognition sequences may produce the same sticky ends. BamHI (recognition sequence GGATCC) and BglII (AGATCT) are examples-both produce GATC sticky ends . The same sticky end is also produced by Sau3A, which recognizes only the tetranucleotide GATC. Fragments of DNA produced by cleavage with either of these enzymes can be joined to each other, as each fragment carries a complementary sticky end.

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