Restriction modification system

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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.

Contents

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. 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 Salvatore Luria and Mary Human in 1952 and 1953. [1] [2] They found that a 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). [3] In 1953, Jean Weigle and Giuseppe Bertani reported similar examples of host-controlled modification using different bacteriophage system. [4] Later work by Daisy Roulland-Dussoix and Werner Arber in 1962 [5] 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. [6] 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.[ citation needed ]

Types

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.[ citation needed ]

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. [7] 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.[ citation needed ]

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 selfish genetic elements, forcing their maintenance on the cell through postsegregational cell killing. [15]

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. [17] 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. [18] [19] [20] 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. [21] [22]

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

Related Research Articles

A restriction enzyme, restriction endonuclease, REase, ENase orrestrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone of the DNA double helix.

<i>Neisseria</i> Genus of bacteria

Neisseria is a large genus of bacteria that colonize the mucosal surfaces of many animals. Of the 11 species that colonize humans, only two are pathogens, N. meningitidis and N. gonorrhoeae.

<span class="mw-page-title-main">Salvador Luria</span> Italian American microbiologist (1912–1991)

Salvador Edward Luria was an Italian microbiologist, later a naturalized U.S. citizen. He won the Nobel Prize in Physiology or Medicine in 1969, with Max Delbrück and Alfred Hershey, for their discoveries on the replication mechanism and the genetic structure of viruses. Salvador Luria also showed that bacterial resistance to viruses (phages) is genetically inherited.

In molecular biology, endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically, while many, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences. Endonucleases differ from exonucleases, which cleave the ends of recognition sequences instead of the middle (endo) portion. Some enzymes known as "exo-endonucleases", however, are not limited to either nuclease function, displaying qualities that are both endo- and exo-like. Evidence suggests that endonuclease activity experiences a lag compared to exonuclease activity.

<i>Neisseria meningitidis</i> Species of bacterium that can cause meningitis

Neisseria meningitidis, often referred to as the meningococcus, is a Gram-negative bacterium that can cause meningitis and other forms of meningococcal disease such as meningococcemia, a life-threatening sepsis. The bacterium is referred to as a coccus because it is round, and more specifically a diplococcus because of its tendency to form pairs.

<i>Nla</i>III Enzyme

NlaIII is a type II restriction enzyme isolated from Neisseria lactamica. As part of the restriction modification system, NlaIII is able to prevent foreign DNA from integrating into the host genome by cutting double stranded DNA into fragments at specific sequences. This results in further degradation of the fragmented foreign DNA and prevents it from infecting the host genome.

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. Alongside CRISPR/Cas9 and TALEN, ZFN is a prominent tool in the field of genome editing.

<i>Hin</i>dIII Enzyme

HindIII (pronounced "Hin D Three") is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that cleaves the DNA palindromic sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis.

<span class="mw-page-title-main">Homing endonuclease</span>

The homing endonucleases are a collection of endonucleases encoded either as freestanding genes within introns, as fusions with host proteins, or as self-splicing inteins. They catalyze the hydrolysis of genomic DNA within the cells that synthesize them, but do so at very few, or even singular, locations. Repair of the hydrolyzed DNA by the host cell frequently results in the gene encoding the homing endonuclease having been copied into the cleavage site, hence the term 'homing' to describe the movement of these genes. Homing endonucleases can thereby transmit their genes horizontally within a host population, increasing their allele frequency at greater than Mendelian rates.

PstI is a type II restriction endonuclease isolated from the Gram negative species, Providencia stuartii.

<span class="mw-page-title-main">Molecular cloning</span> Set of methods in molecular biology


Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

<span class="mw-page-title-main">Genome editing</span> Type of genetic engineering

Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site-specific locations. The basic mechanism involved in genetic manipulations through programmable nucleases is the recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases, and the repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining (NHEJ).

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

<span class="mw-page-title-main">Cas9</span> Microbial protein found in Streptococcus pyogenes M1 GAS

Cas9 is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 being awarded to Emmanuelle Charpentier and Jennifer Doudna.

<span class="mw-page-title-main">Daisy Roulland-Dussoix</span> Swiss microbiologist and molecular biologist (1936–2014)

Daisy Roulland-Dussoix was a Swiss molecular microbiologist. She was one of the discoverers of restriction enzymes during her doctoral studies. There is controversy over whether she should have received the 1978 Nobel prize in Physiology and Medicine, which was awarded to Hamilton O. Smith, Daniel Nathans, and Werner Arber.

<span class="mw-page-title-main">Phasevarion</span>

In bacteria, phasevarions mediate a coordinated change in the expression of multiple genes or proteins. This occurs via phase variation of a single DNA methyltransferase. Phase variation of methyltransferase expression results in differential methylation throughout the bacterial genome, leading to variable expression of multiple genes through epigenetic mechanisms.

Since antiretroviral therapy requires a lifelong treatment regimen, research to find more permanent cures for HIV infection is currently underway. It is possible to synthesize zinc finger nucleotides with zinc finger components that selectively bind to specific portions of DNA. Conceptually, targeting and editing could focus on host cellular co-receptors for HIV or on proviral HIV DNA.

References

  1. ↑ Luria SE, Human ML (1952). "A nonhereditary, host-induced variation of bacterial viruses". J. Bacteriol. 64 (4): 557–69. doi:10.1128/JB.64.4.557-569.1952. PMC   169391 . PMID   12999684.
  2. ↑ Luria SE (1953). "Host-induced modifications of viruses". Cold Spring Harb. Symp. Quant. Biol. 18: 237–44. doi:10.1101/sqb.1953.018.01.034. PMID   13168990.
  3. 1 2 Salvator E Luria. A Slot Machine, A Broken Test Tube: An Autobiography. Harper & Row, New York: 1984. Pp. 228. ISBN   0-06-015260-5 (USA and Canada)
  4. ↑ BERTANI G, WEIGLE JJ (1953). "Host controlled variation in bacterial viruses". J. Bacteriol. 65 (2): 113–21. doi:10.1128/JB.65.2.113-121.1953. PMC   169650 . PMID   13034700.
  5. ↑ DUSSOIX D, ARBER W (1962). "Host specificity of DNA produced by Escherichia coli. II. Control over acceptance of DNA from infecting phage lambda". J. Mol. Biol. 5: 37–49. doi:10.1016/S0022-2836(62)80059-X. PMID   13888713.
  6. ↑ Nathans D, Smith HO (1975). "Restriction endonucleases in the analysis and restructuring of dna molecules". Annu. Rev. Biochem. 44: 273–93. doi:10.1146/annurev.bi.44.070175.001421. PMID   166604.
  7. ↑ Rodic, A; Blagojevic, B; Zdobnov, E; Djordjevic, M; Djordjevic, M (24 February 2017). "Understanding key features of bacterial restriction-modification systems through quantitative modeling". BMC systems biology. 11 (Suppl 1): 377. doi:10.1186/s12918-016-0377-x. PMID   28466789.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  8. ↑ Wilson G (1991). "Organization of Restriction-Modification Systems". Nucleic Acids Research . 19 (10): 2539–2566. doi:10.1093/nar/19.10.2539. PMC   328170 . PMID   2041731.
  9. ↑ Wilson G (1991). "Restriction and Modification Systems". Annual Review of Genetics. 25: 585–627. doi:10.1146/annurev.ge.25.120191.003101. PMID   1812816.
  10. ↑ Loenen WA (2013). "The other face of restriction: modification-dependent enzymes". Nucleic Acids Research. 42 (1): 56–69. doi:10.1093/nar/gkt747. PMC   3874153 . PMID   23990325.
  11. 1 2 3 Budroni S, Siena E, Dunning Hotopp JC, Seib KL, Serruto D, Nofroni C, Comanducci M, Riley DR, Daugherty SC, Angiuoli SV, Covacci A, Pizza M, Rappuoli R, Moxon ER, Tettelin H, Medini D (2011). "Neisseria meningitidis is structured in clades associated with restriction modification systems that modulate homologous recombination". Proc. Natl. Acad. Sci. U.S.A. 108 (11): 4494–9. Bibcode:2011PNAS..108.4494B. doi: 10.1073/pnas.1019751108 . PMC   3060241 . PMID   21368196.
  12. ↑ Claus H, Friedrich A, Frosch M, Vogel U (2000). "Differential distribution of novel restriction-modification systems in clonal lineages of Neisseria meningitidis". J. Bacteriol. 182 (5): 1296–303. doi:10.1128/jb.182.5.1296-1303.2000. PMC   94415 . PMID   10671450.
  13. ↑ Ambur OH, Frye SA, Nilsen M, Hovland E, Tønjum T (2012). "Restriction and sequence alterations affect DNA uptake sequence-dependent transformation in Neisseria meningitidis". PLOS ONE. 7 (7): e39742. Bibcode:2012PLoSO...739742A. doi: 10.1371/journal.pone.0039742 . PMC   3388099 . PMID   22768309.
  14. ↑ Caugant DA, Maiden MC (2009). "Meningococcal carriage and disease--population biology and evolution". Vaccine. 27 Suppl 2 (4): B64–70. doi:10.1016/j.vaccine.2009.04.061. PMC   2719693 . PMID   19464092.
  15. ↑ Kusano K (1995). "Restriction-Modification Systems as Genomic Parasites in Competition for Specific Sequences". Proceedings of the National Academy of Sciences of the United States of America. 92 (24): 11095–11099. Bibcode:1995PNAS...9211095K. doi: 10.1073/pnas.92.24.11095 . PMC   40578 . PMID   7479944.
  16. ↑ Oliveira, Pedro H.; Touchon, Marie; Rocha, Eduardo P.C. (2016-05-17). "Regulation of genetic flux between bacteria by restriction–modification systems". Proceedings of the National Academy of Sciences. 113 (20): 5658–5663. Bibcode:2016PNAS..113.5658O. doi: 10.1073/pnas.1603257113 . ISSN   0027-8424. PMC   4878467 . PMID   27140615.
  17. ↑ 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.
  18. ↑ 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.
  19. ↑ Schiffer JT, 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.
  20. ↑ Manjunath N, 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.
  21. ↑ Stetson DB, Ko JS, Heidmann T, Medzhitov R (2008). "Trex1 prevents cell intrinsic initiation of autoimmunity". Cell. 134 (4): 587–598. doi:10.1016/j.cell.2008.06.032. PMC   2626626 . PMID   18724932.
  22. ↑ 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. PMC   2483505 . PMID   18396111.
  23. ↑ 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.
  24. ↑ 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. PMC   4084652 . PMID   24597865.
  25. 1 2 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. PMC   4176335 . PMID   25120263.
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