Biochemistry - The Chemical Reactions of Living Cells, Volume 3 - D. Metzler 1980

Biochemical Genetics and the Synthesis of Nucleic Acids and Proteins
DNA Restriction and Modification
Restriction Endonucleases

A relentless arms race is constantly underway between Viruses and Bacteria, with both the attacking phage and the host bacterium employing a variety of offensive and defensive mechanisms. For instance, upon invading a bacterial Cell, many viruses not only shut down host DNA Synthesis but also degrade the DNA through the action of specific Enzymes—both endonucleases and exonucleases—whose synthesis is encoded within the viral genome [211–214]. Conversely, host bacteria utilize their own defensive arsenal, frequently modifying viral DNA to protect themselves against its destructive effects¹⁾. Thus, enzymes determined by T-even phages convert cytidine monophosphate into 5'-hydroxymethyl-CMP, with these modified NUCLEOTIDES being incorporated into the viral DNA [212]. Furthermore, the newly formed hydroxyl group can be glucosylated to varying degrees (Chap. 2, Sec. D, 8).

¹⁾ Interestingly, bacteria evolve in a way that avoids interfering with phage propagation. Many mutants (grov), however, block the growth of phages such as λ, T3, T4, and T7 while continuing to grow normally themselves. The fact that such bacteria do not revert to the wild type highlights the vital importance of coexistence with the phage for the bacterium.

Bacteria frequently digest and degrade the DNA of invading viruses or DNA introduced into The Cell through conjugation with an incompatible bacterial strain. Investigations into this fascinating phenomenon, known as restriction, revealed that the DNA of viruses capable of replicating only in specific host Cells is somehow marked at specific sites. In many cases, these markers are methyl groups. It was found that properly methylated DNA is not cleaved by the bacterium, whereas unmethylated DNA is degraded by a highly specific endonuclease precisely at the sites where methylation normally occurs. Each bacterial species (and frequently even individual strains within a given species) possesses its own unique restriction enzymes. Restriction enzymes exhibit an exceptionally high degree of Specificity, often cleaving DNA at only a few points (or adjacent to them) characterized by a unique base sequence. Currently, about 45 such enzymes with distinct specificities have been isolated.

Restriction Endonucleases determined by the E. coli chromosome are large Proteins with molecular weights on the order of 300,000–400,000, consisting of Three types of polypeptide chains. They clearly bind to specific sites and non-specifically degrade adjacent regions. Their activity requires the presence of ATP, Mg²⁺ ions, and S-adenosylmethionine. A unique feature of these proteins is their ability to induce the Hydrolysis of unusually large amounts of ATP [215]. The Significance of all these properties of restriction enzymes remains unclear. The second Class of restriction enzymes comprises relatively small monomeric or dimeric proteins with molecular weights of 50,000–100,000. The target sites for these enzymes are typically nucleotide sequences with local twofold rotational Symmetry [217]. For example, the following Cleavage sites have been identified for two restriction endonucleases determined by the E. coli R-factor plasmid and a restriction enzyme from Hemophilus influenzae (in the scheme below, arrows indicate cleavage sites, asterisks denote methylation sites, and dots represent the local twofold axis of symmetry):

E. coli R-factor (Eco RI)

E. coli R-factor (Eco RII)

H. influenzae (Hind III)

In many cases, restriction enzymes introduce breaks in both strands at positions symmetrically disposed relative to the twofold axis of symmetry. This is precisely what would be expected if a dimeric enzyme binds to the major or minor groove of The Double Helix, with each Active Site interacting with one of the polynucleotide chains.