Restriction modification system

The restriction modification system (RM system) is found in bacteria and other prokaryotic organisms, and provides a defense against foreign DNA, such as that borne by bacteriophages.. The restriction modification system (RM system) is found in bacteria and other prokaryotic organisms, and provides a defense against foreign DNA, such as that borne by bacteriophages.. Bacteria have restriction enzymes, also called restriction endonucleases, which cleave double stranded DNA at specific points into fragments, which are then degraded further by other endonucleases. This prevents infection by effectively destroying the foreign DNA introduced by an infectious agent (such as a bacteriophage). Approximately one-quarter of known bacteria possess RM systems and of those about one-half have more than one type of system. As the sequences recognized by the restriction enzymes are very short, the bacterium itself will almost certainly contain some within its genome. In order to prevent destruction of its own DNA by the restriction enzymes, methyl groups are added. These modifications must not interfere with the DNA base-pairing, and therefore, usually only a few specific bases are modified on each strand. Endonucleases cleave internal/non-terminal phosphodiester bonds. Restriction endonucleases cleave internal phosphodiester bonds only after recognising specific sequences in DNA which are usually 4-6 base pairs long, and often palindromic. The RM system was first discovered by Salvatore Luria and Mary Human in 1952 and 1953. They found that bacteriophage growing within an infected bacterium could be modified, so that upon their release and re-infection of a related bacterium the bacteriophage’s growth is restricted (inhibited) (also described by Luria in his autobiography on pages 45 and 99 in 1984). In 1953, Jean Weigle and Giuseppe Bertani reported similar examples of host-controlled modification using different bacteriophage system. Later work by Daisy Roulland-Dussoix and Werner Arber in 1962 and many other subsequent workers led to the understanding that restriction was due to attack and breakdown of the modified bacteriophage’s DNA by specific enzymes of the recipient bacteria. Further work by Hamilton O. Smith isolated HinDII, the first of the class of enzymes now known as restriction enzymes, while Daniel Nathans showed that it can be used for restriction mapping. When these enzymes were isolated in the laboratory they could be used for controlled manipulation of DNA, thus providing the foundation for the development of genetic engineering. Werner Arber, Daniel Nathans, and Hamilton Smith were awarded the Nobel Prize in Physiology or Medicine in 1978 for their work on restriction-modification. There are four categories of restriction modification systems: type I, type II, type III and type IV, all with restriction enzyme activity and a methylase activity (except for type IV that has no methylase activity). They were named in the order of discovery, although the type II system is the most common. Type I systems are the most complex, consisting of three polypeptides: R (restriction), M (modification), and S (specificity). The resulting complex can both cleave and methylate DNA. Both reactions require ATP, and cleavage often occurs a considerable distance from the recognition site. The S subunit determines the specificity of both restriction and methylation. Cleavage occurs at variable distances from the recognition sequence, so discrete bands are not easily visualized by gel electrophoresis. Type II systems are the simplest and the most prevalent. Instead of working as a complex, the methyltransferase and endonuclease are encoded as two separate proteins and act independently (there is no specificity protein). Both proteins recognize the same recognition site, and therefore compete for activity. The methyltransferase acts as a monomer, methylating the duplex one strand at a time. The endonuclease acts as a homodimer, which facilitates the cleavage of both strands. Cleavage occurs at a defined position close to or within the recognition sequence, thus producing discrete fragments during gel electrophoresis. For this reason, Type II systems are used in labs for DNA analysis and gene cloning. Type III systems have R (res) and M (mod) proteins that form a complex of modification and cleavage. The M protein, however, can methylate on its own. Methylation also only occurs on one strand of the DNA unlike most other known mechanisms. The heterodimer formed by the R and M proteins competes with itself by modifying and restricting the same reaction. This results in incomplete digestion.