Restriction modification system
The restriction modification system (RM system) is used by bacteria, and perhaps other prokaryotic organisms, to protect themselves from foreign DNA, such as the one borne by bacteriophages. This phenomenon was first noticed in the 1950s. Certain bacteria strains were found to inhibit (restrict) the growth of viruses grown in previous strains. This effect was attributed to sequence-specific restriction enzymes.
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.
Types of restriction modification system
There are five kinds of restriction modification system: type I, type II, type IIS, type III and type IV, all with restriction enzyme activity and a 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 and M 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.
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.
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.
(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.
(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 specificity of 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 nonb-specific to the mutant sequence.
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. Research is on REases and ZFN that can cleave the DNA of various human viruses, including HSV-2, high-risk HPVs and HIV-1, with the ultimate goal of inducing target mutagenesis and aberrations of human-infecting viruses. Interestingly, the human genome already contains remnants of retroviral genomes that have been inactivated and harnessed for self-gain. Indeed, the mechanisms for silencing active L1 genomic retroelements by the three prime repair exonuclease 1 (TREX1) and excision repair cross complementing 1(ERCC) appear to mimic the action of RM-systems in bacteria, and the non-homologous end-joining (NHEJ) that follows the use of ZFN without a repair template.
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).) 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 
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. 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. 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.
- Wilson, G., "Organization of Restriction-Modification Systems,"Nucleic Acids Research (1991), Vol 19, pg2539-2566.
- Wilson, G., "Restriction and Modification Systems," Annual Review of Genetics (1991), 25:585-627.
- Wayengera M (2003). "HIV and Gene Therapy: The proposed [R-M enzymatic] model for a gene therapy against HIV.". Makerere Med J. 38: 28–30.
- Wayengera M, Kajumbula H, Byarugaba W (2007). "Frequency and site mapping of HIV-1/SIVcpz, HIV-2/SIVsmm and Other SIV gene sequence cleavage by various bacteria restriction enzymes: Precursors for a novel HIV inhibitory product". Afr J Biotechnol 6 (10): 1225–1232.
- Schiffer JT1, Aubert M, Weber ND, Mintzer E, Stone D, Jerome KR (2012). "Targeted DNA mutagenesis for the cure of chronic viral infections". Journal of Virology 86 (17): 8920–36. doi:10.1128/JVI.00052-12. PMC 3416169. PMID 22718830.
- Manjunath N1, Yi G, Dang Y, Shankar P (2013). "Newer gene editing technologies toward HIV gene therapy". Viruses 5 (11): 2748–66. doi:10.3390/v5112748. PMC 3856413. PMID 24284874.
- Stetson DB, Ko JS, Heidmann T, Medzhitov R (2008). "Trex1 prevents cell intrinsic initiation of autoimmunity". Cell 134: 587–598. doi:10.1016/j.cell.2008.06.032. PMC 2626626. PMID 18724932.
- Gasior SL, Roy-Engel AM, Deininger PL (2008). "ERCC1/XPF limits L1 retrotransposition". DNA Repair 7 (6): 983–989. doi:10.1016/j.dnarep.2008.02.006. PMID 18396111.
- Kim YG, Cha J, Chandrasegaran S (February 1996). "Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain". Proc. Natl. Acad. Sci. U.S.A. 93 (3): 1156–60. Bibcode:1996PNAS...93.1156K. doi:10.1073/pnas.93.3.1156. PMC 40048. PMID 8577732.
- Tebas P, Stein D, Tang WW, Frank I, Wang SQ, Lee G et al. (2014). "Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV". N Engl J Med 370 (10): 901–910. doi:10.1056/NEJMoa1300662. PMID 24597865.
- Oliveira, PH; Touchon, M; Rocha, EPC (2014). "The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts". Nucleic Acids Res 42 (16): 10618–10631. doi:10.1093/nar/gku734.