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.

Bacteria have

Endonucleases cleave internal/non-terminal phosphodiester bonds. They do so only after recognising specific sequences in DNA which are usually 4–6 base pairs long, and often palindromic.

History

The RM system was first discovered by

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.^{[citation needed]}

Type II systems are the simplest and the most prevalent.

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.^[8][9]

Type IV systems are not true RM systems because they only contain a restriction enzyme and not a methylase. Unlike the other types, type IV restriction enzymes recognize and cut only modified DNA.^[10]

Function

Neisseria meningitidis has multiple type II restriction endonuclease systems that are employed in natural genetic transformation. Natural genetic transformation is a process by which a recipient bacterial cell can take up DNA from a neighboring donor bacterial cell and integrate this DNA into its genome by recombination. Although early work on restriction modification systems focused on the benefit to bacteria of protecting themselves against invading bacteriophage DNA or other foreign DNA, it is now known that these systems can also be used to restrict DNA introduced by natural transformation from other members of the same, or related species.^{[citation needed]}

In the pathogenic bacterium Neisseria meningitidis (meningococci), competence for transformation is a highly evolved and complex process where multiple proteins at the bacterial surface, in the membranes and in the cytoplasm interact with the incoming transforming DNA. Restriction-modification systems are abundant in the genus Neisseria. N. meningitidis has multiple type II restriction endonuclease systems.^[11] The restriction modification systems in N. meningitidis vary in specificity between different clades.^[11][12] This specificity provides an efficient barrier against DNA exchange between clades.^[11] Luria, on page 99 of his autobiography,^[3] referred to such a restriction behavior as "an extreme instance of unfriendliness." Restriction-modification appears to be a major driver of sexual isolation and speciation in the meningococci.^[13] Caugant and Maiden^[14] suggested that restriction-modification systems in meningococci may act to allow genetic exchange among very close relatives while reducing (but not completely preventing) genetic exchange among meningococci belonging to different clonal complexes and related species.^{[citation needed]}

RM systems can also act as

Some viruses have evolved ways of subverting the restriction modification system, usually by modifying their own DNA, by adding methyl or glycosyl groups to it, thus blocking the restriction enzymes. Other viruses, such as bacteriophages T3 and T7, encode proteins that inhibit the restriction enzymes.^{[citation needed]}

To counteract these viruses, some bacteria have evolved restriction systems which only recognize and cleave modified DNA, but do not act upon the host's unmodified DNA. Some prokaryotes have developed multiple types of restriction modification systems.^{[citation needed]}

R-M systems are more abundant in promiscuous species, wherein they establish preferential paths of genetic exchange within and between lineages with cognate R-M systems.^[16] Because the repertoire and/or specificity of R-M systems in bacterial lineages vary quickly, the preferential fluxes of genetic transfer within species are expected to constantly change, producing time-dependent networks of gene transfer.^{[citation needed]}

Applications

Molecular biology

(a) Cloning: RM systems can be cloned into plasmids and selected because of the resistance provided by the methylation enzyme. Once the plasmid begins to replicate, the methylation enzyme will be produced and methylate the plasmid DNA, protecting it from a specific restriction enzyme.^{[citation needed]}

(b) Restriction fragment length polymorphisms: Restriction enzymes are also used to analyse the composition of DNA in regard to presence or absence of mutations that affect the REase cleavage specificity. When wild-type and mutants are analysed by digestion with different REases, the gel-electrophoretic products vary in length, largely because mutant genes will not be cleaved in a similar pattern as wild-type for presence of mutations that render the REases non-specific to the mutant sequence.^{[citation needed]}

Gene therapy

The bacteria R-M system has been proposed as a model for devising human anti-viral gene or genomic vaccines and therapies since the RM system serves an innate defense-role in bacteria by restricting tropism of bacteriophages.

A major advance is the creation of artificial restriction enzymes created by linking the FokI DNA cleavage domain with an array of DNA binding proteins or zinc finger arrays, denoted now as zinc finger nucleases (ZFN).[23] ZFNs are a powerful tool for host genome editing due to their enhanced sequence specificity. ZFN work in pairs, their dimerization being mediated in-situ through the FoKI domain. Each zinc finger array (ZFA) is capable of recognizing 9–12 base-pairs, making for 18–24 for the pair. A 5–7 bp spacer between the cleavage sites further enhances the specificity of ZFN, making them a safe and more precise tool that can be applied in humans. A recent Phase I clinical trial of ZFN for the targeted abolition of the CCR5 co-receptor for HIV-1 has been undertaken.^[24]

Their relation with mobile genetic elements (MGEs)

R-M systems are major players in the co-evolutionary interaction between mobile genetic elements (MGEs) and their hosts.^[25] Genes encoding R-M systems have been reported to move between prokaryotic genomes within MGEs such as plasmids, prophages, insertion sequences/transposons, integrative conjugative elements (ICEs) and integrons. However, it was recently found that there are relatively few R-M systems in plasmids, some in prophages, and practically none in phages. On the other hand, all these MGEs encode a large number of solitary R-M genes, notably MTases.^[25] In light of this, it is likely that R-M mobility may be less dependent on MGEs and more dependent, for example, on the existence of small genomic integration hotspots. It is also possible that R-M systems frequently exploit other mechanisms such as natural transformation, vesicles, nanotubes, gene transfer agents or generalized transduction in order to move between genomes.^{[citation needed]}

See also

• Methylation
• Restriction enzyme

References