Editing Restriction enzyme (section)

== Types ==
Naturally occurring restriction endonucleases are categorized into five groups (Types I, II, III, IV, and V) based on their composition and [[enzyme cofactor]] requirements, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence.<ref name="pmid8336674">{{cite journal | vauthors = Bickle TA, Krüger DH | title = Biology of DNA restriction | journal = Microbiological Reviews | volume = 57 | issue = 2 | pages = 434–50 | date = June 1993 | pmid = 8336674 | pmc = 372918 | doi =  10.1128/MMBR.57.2.434-450.1993}}</ref><ref name="pmid4949033">{{cite journal | vauthors = Boyer HW | title = DNA restriction and modification mechanisms in bacteria | journal = Annual Review of Microbiology | volume = 25 | pages = 153–76 | year = 1971 | pmid = 4949033 | doi = 10.1146/annurev.mi.25.100171.001101 }}</ref><ref name="pmid6267988">{{cite journal | vauthors = Yuan R | title = Structure and mechanism of multifunctional restriction endonucleases | journal = Annual Review of Biochemistry | volume = 50 | pages = 285–319 | year = 1981 | pmid = 6267988 | doi = 10.1146/annurev.bi.50.070181.001441 }}</ref>  DNA sequence analysis of restriction enzymes however show great variations, indicating that there are more than four types.<ref name="neb"/> All types of enzymes recognize specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific fragments with terminal 5'-phosphates. They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements,<ref name="pmid15121719">{{cite journal | vauthors = Sistla S, Rao DN | title = S-Adenosyl-L-methionine-dependent restriction enzymes | journal = Critical Reviews in Biochemistry and Molecular Biology | volume = 39 | issue = 1 | pages = 1–19 | year = 2004 | pmid = 15121719 | doi = 10.1080/10409230490440532 | s2cid = 1929381 }}</ref><ref name="PUB00035707">{{cite journal | vauthors = Williams RJ | title = Restriction endonucleases: classification, properties, and applications | journal = Molecular Biotechnology | volume = 23 | issue = 3 | pages = 225–43 | date = March 2003 | pmid = 12665693 | doi = 10.1385/MB:23:3:225 | s2cid = 29672999 }}</ref> as summarised below:

* Type I enzymes ({{EC number|3.1.21.3}}) cleave at sites remote from a recognition site; require both [[Adenosine triphosphate|ATP]] and [[S-Adenosyl methionine|S-adenosyl-L-methionine]] to function; multifunctional protein with both restriction digestion and methylase ({{EC number|2.1.1.72}}) activities.
* Type II enzymes ({{EC number|3.1.21.4}}) cleave within or at short specific distances from a recognition site; most require [[magnesium]]; single function (restriction digestion) enzymes independent of methylase.
* Type III enzymes ({{EC number|3.1.21.5}}) cleave at sites a short distance from a recognition site; require ATP (but do not hydrolyse it); S-adenosyl-L-methionine stimulates the reaction but is not required; exist as part of a complex with a modification methylase ({{EC number|2.1.1.72}}).
* Type IV enzymes target modified DNA, e.g. methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA
* Type V enzymes utilize [[Guide RNA|guide RNAs (gRNAs)]]

=== Type l ===
Type I restriction enzymes were the first to be identified and were first identified in two different strains (K-12 and B) of ''[[Escherichia coli|E. coli]]''.<ref name="pmid10839821">{{cite journal | vauthors = Murray NE | title = Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle) | journal = Microbiology and Molecular Biology Reviews | volume = 64 | issue = 2 | pages = 412–34 | date = June 2000 | pmid = 10839821 | pmc = 98998 | doi = 10.1128/MMBR.64.2.412-434.2000 }}</ref> These enzymes cut at a site that differs, and is a random distance (at least 1000 bp) away, from their recognition site. Cleavage at these random sites follows a process of DNA translocation, which shows that these enzymes are also molecular motors. The recognition site is asymmetrical and is composed of two specific portions—one containing 3–4 nucleotides, and another containing 4–5 nucleotides—separated by a non-specific spacer of about 6–8 nucleotides. These enzymes are multifunctional and are capable of both restriction digestion and modification activities, depending upon the methylation status of the target DNA. The cofactors [[S-Adenosyl methionine]] (AdoMet), hydrolyzed adenosine triphosphate ([[Adenosine triphosphate|ATP]]), and [[magnesium]] (Mg<sup>2+</sup>) [[ion]]s, are required for their full activity. Type I restriction enzymes possess three subunits called HsdR, HsdM, and HsdS; HsdR is required for restriction digestion; HsdM is necessary for adding [[methyl]] groups to host DNA (methyltransferase activity), and HsdS is important for specificity of the recognition (DNA-binding) site in addition to both restriction digestion (DNA cleavage) and modification (DNA methyltransferase) activity.<ref name="pmid8336674"/><ref name="pmid10839821"/>

=== Type II ===
{{Infobox protein family
| Name = Type II site-specific deoxyribonuclease-like
|Symbol= Restrct_endonuc-II-like
| EC_number = 3.1.21.4
| CAS_number = 9075-08-5
| IUBMB_EC_number = 3/1/21/4
| footnote = GO:0009036
| image = 1QPS.png
| width =
|InterPro=IPR011335
|Pfam_clan=CL0236
|SCOP=1wte
| caption = Structure of the [[protein dimer|homodimeric]] restriction enzyme [[EcoRI]] (cyan and green cartoon diagram) bound to double stranded [[DNA]] (brown tubes).<ref name="pdb_1qps">{{PDB|1qps}} {{cite book |vauthors=Gigorescu A, Morvath M, Wilkosz PA, Chandrasekhar K, Rosenberg JM | editor = Alfred M. Pingoud | title = Restriction Endonucleases (Nucleic Acids and Molecular Biology, Volume 14) | publisher = Springer | location = Berlin | year = 2004 | chapter = The integration of recognition and cleavage: X-ray structures of pre-transition state complex, post-reactive complex, and the DNA-free endonuclease | pages = 137–178 | isbn = 3-540-20502-0 }}</ref> Two catalytic [[magnesium]] ions (one from each [[monomer]]) are shown as magenta spheres and are adjacent to the cleaved sites in the DNA made by the enzyme (depicted as gaps in the DNA backbone).
}}
Typical type II restriction enzymes differ from type I restriction enzymes in several ways. They form [[homodimer]]s, with recognition sites that are usually undivided and palindromic and 4–8 nucleotides in length. They recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their activity—they usually require only Mg<sup>2+</sup> as a cofactor.<ref name="pmid11557805"/>  These enzymes cleave the phosphodiester bond of double helix DNA. It can either cleave at the center of both strands to yield a blunt end, or at a staggered position leaving overhangs called sticky ends.<ref>{{Cite book|title=Fundamental Laboratory Approaches for Biochemistry and Biotechnology| vauthors = Ninfa JP, Balou DP, Benore M |publisher=John Wiley & Sons|year=2010|isbn=978-0-470-08766-4|location=Hoboken, NJ|pages=341}}</ref> These are the most commonly available and used restriction enzymes. In the 1990s and early 2000s, new enzymes from this family were discovered that did not follow all the classical criteria of this enzyme class, and new subfamily [[nomenclature]] was developed to divide this large family into subcategories based on deviations from typical characteristics of type II enzymes.<ref name="pmid11557805"/> These subgroups are defined using a letter suffix.

Type IIB restriction enzymes (e.g., BcgI and BplI) are [[multimer]]s, containing more than one subunit.<ref name="pmid11557805"/> They cleave DNA on both sides of their recognition to cut out the recognition site. They require both AdoMet and Mg<sup>2+</sup> cofactors. Type IIE restriction endonucleases (e.g., NaeI) cleave DNA following interaction with two copies of their recognition sequence.<ref name="pmid11557805"/> One recognition site acts as the target for cleavage, while the other acts as an [[Allosteric regulation|allosteric effector]] that speeds up or improves the efficiency of enzyme cleavage. Similar to type IIE enzymes, type IIF restriction endonucleases (e.g. NgoMIV) interact with two copies of their recognition sequence but cleave both sequences at the same time.<ref name="pmid11557805"/> Type IIG restriction endonucleases (e.g., RM.Eco57I) do have a single subunit, like classical Type II restriction enzymes, but require the cofactor AdoMet to be active.<ref name="pmid11557805"/> Type IIM restriction endonucleases, such as [[DpnI]], are able to recognize and cut methylated DNA.<ref name="pmid11557805"/><ref>{{cite journal | vauthors = Siwek W, Czapinska H, Bochtler M, Bujnicki JM, Skowronek K | title = Crystal structure and mechanism of action of the N6-methyladenine-dependent type IIM restriction endonuclease R.DpnI | journal = Nucleic Acids Research | volume = 40 | issue = 15 | pages = 7563–72 | date = August 2012 | pmid = 22610857 | pmc = 3424567 | doi = 10.1093/nar/gks428 }}</ref><ref>{{cite journal | vauthors = Mierzejewska K, Siwek W, Czapinska H, Kaus-Drobek M, Radlinska M, Skowronek K, Bujnicki JM, Dadlez M, Bochtler M | display-authors = 6 | title = Structural basis of the methylation specificity of R.DpnI | journal = Nucleic Acids Research | volume = 42 | issue = 13 | pages = 8745–54 | date = July 2014 | pmid = 24966351 | pmc = 4117772 | doi = 10.1093/nar/gku546 }}</ref> Type IIS restriction endonucleases (e.g. FokI) cleave DNA at a defined distance from their non-palindromic asymmetric recognition sites;<ref name="pmid11557805"/> this characteristic is widely used to perform in-vitro cloning techniques such as [[Golden Gate Cloning|Golden Gate cloning]]. These enzymes may function as [[protein dimer|dimer]]s. Similarly, Type IIT restriction enzymes (e.g., Bpu10I and BslI) are composed of two different subunits. Some recognize palindromic sequences while others have asymmetric recognition sites.<ref name="pmid11557805"/>
{{Further|BsuBI/PstI restriction endonuclease}}

=== Type III ===
Type III restriction enzymes (e.g., EcoP15) recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20–30 base pairs after the recognition site.<ref name="pmid11557806">{{cite journal | vauthors = Dryden DT, Murray NE, Rao DN | title = Nucleoside triphosphate-dependent restriction enzymes | journal = Nucleic Acids Research | volume = 29 | issue = 18 | pages = 3728–41 | date = September 2001 | pmid = 11557806 | pmc = 55918 | doi = 10.1093/nar/29.18.3728 }}</ref> These enzymes contain more than one subunit and require AdoMet and ATP cofactors for their roles in DNA methylation and restriction digestion, respectively.<ref name="pmid1734285">{{cite journal | vauthors = Meisel A, Bickle TA, Krüger DH, Schroeder C | title = Type III restriction enzymes need two inversely oriented recognition sites for DNA cleavage | journal = Nature | volume = 355 | issue = 6359 | pages = 467–9 | date = January 1992 | pmid = 1734285 | doi = 10.1038/355467a0 | bibcode = 1992Natur.355..467M | s2cid = 4354056 }}</ref> They are components of [[Prokaryote|prokaryotic]] DNA restriction-modification [[Mechanism (biology)|mechanisms]] that protect the organism against invading foreign DNA. Type III enzymes are hetero-oligomeric, multifunctional [[protein]]s composed of two subunits, Res ({{uniProt|P08764}}) and Mod ({{uniProt|P08763}}). The Mod subunit recognises the DNA sequence specific for the system and is a modification [[methyltransferase]]; as such, it is functionally equivalent to the M and S subunits of type I restriction endonuclease. Res is required for restriction digestion, although it has no [[enzyme|enzymatic]] activity on its own. Type III enzymes recognise short 5–6 bp-long asymmetric DNA sequences and cleave 25–27 bp [[Upstream and downstream (DNA)|downstream]] to leave short, single-stranded 5' protrusions. They require the presence of two inversely oriented unmethylated recognition sites for restriction digestion to occur. These enzymes [[DNA methylation|methylate]] only one strand of the DNA, at the N-6 position of adenine residues, so newly replicated DNA will have only one strand methylated, which is sufficient to protect against restriction digestion. Type III enzymes belong to the beta-subfamily of [[DNA methyltransferase|N6 adenine methyltransferases]], containing the nine [[protein motif|motif]]s that characterise this family, including [[sequence motif|motif]] I, the [[S-adenosyl-L-methionine|AdoMet]] binding pocket (FXGXG), and motif IV, the [[catalytic]] region (S/D/N (PP) Y/F).<ref name="pmid15121719"/><ref name="pmid12595133">{{cite journal | vauthors = Bourniquel AA, Bickle TA | title = Complex restriction enzymes: NTP-driven molecular motors | journal = Biochimie | volume = 84 | issue = 11 | pages = 1047–59 | date = November 2002 | pmid = 12595133 | doi = 10.1016/S0300-9084(02)00020-2 }}</ref>

=== Type IV ===
Type IV enzymes recognize modified, typically methylated DNA and are exemplified by the&nbsp;McrBC&nbsp;and Mrr systems of&nbsp;''E. coli''.<ref name="neb">[https://www.neb.com/products/restriction-endonucleases/restriction-endonucleases/types-of-restriction-endonucleases Types of Restriction Endonucleases | NEB<!-- Bot generated title -->]</ref>

===Type V===
Type V restriction enzymes (e.g., the [[cas9]]-gRNA complex from [[CRISPR]]s<ref name=":0" />) utilize guide RNAs to target specific non-palindromic sequences found on invading organisms. They can cut DNA of variable length, provided that a suitable guide RNA is provided. The flexibility and ease of use of these enzymes make them promising for future genetic engineering applications.<ref name=":0">{{cite journal | vauthors = Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P | display-authors = 6 | title = CRISPR provides acquired resistance against viruses in prokaryotes | journal = Science | volume = 315 | issue = 5819 | pages = 1709–12 | date = March 2007 | pmid = 17379808 | doi = 10.1126/science.1138140 | bibcode = 2007Sci...315.1709B | hdl = 20.500.11794/38902 | s2cid = 3888761 | hdl-access = free }}</ref><ref name="pmid20056882">{{cite journal | vauthors = Horvath P, Barrangou R | title = CRISPR/Cas, the immune system of bacteria and archaea | journal = Science | volume = 327 | issue = 5962 | pages = 167–70 | date = January 2010 | pmid = 20056882 | doi = 10.1126/science.1179555 | bibcode = 2010Sci...327..167H | s2cid = 17960960 }}</ref>

=== Artificial restriction enzymes ===
Artificial restriction enzymes can be generated by fusing a natural or engineered [[DNA-binding domain]] to a [[nuclease]] domain (often the cleavage domain of the type IIS restriction enzyme [[FokI]]).<ref name="kim1996">{{cite journal | vauthors = Kim YG, Cha J, Chandrasegaran S | title = Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 93 | issue = 3 | pages = 1156–60 | date = February 1996 | pmid = 8577732 | pmc = 40048 | doi = 10.1073/pnas.93.3.1156 | bibcode = 1996PNAS...93.1156K | doi-access = free }}</ref> Such artificial restriction enzymes can target large DNA sites (up to 36 bp) and can be engineered to bind to desired DNA sequences.<ref name="pmid20717154">{{cite journal | vauthors = Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD | title = Genome editing with engineered zinc finger nucleases | journal = Nature Reviews. Genetics | volume = 11 | issue = 9 | pages = 636–46 | date = September 2010 | pmid = 20717154 | doi = 10.1038/nrg2842 | s2cid = 205484701 }}</ref> [[Zinc finger nuclease]]s are the most commonly used artificial restriction enzymes and are generally used in [[genetic engineering]] applications,<ref name="pmid19404258">{{cite journal | vauthors = Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, Joung JK, Voytas DF | title = High-frequency modification of plant genes using engineered zinc-finger nucleases | journal = Nature | volume = 459 | issue = 7245 | pages = 442–5 | date = May 2009 | pmid = 19404258 | pmc = 2743854 | doi = 10.1038/nature07845 | bibcode = 2009Natur.459..442T }}</ref><ref name="pmid19404259">{{cite journal | vauthors = Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, Rock JM, Wu YY, Katibah GE, Zhifang G, McCaskill D, Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD | display-authors = 6 | title = Precise genome modification in the crop species Zea mays using zinc-finger nucleases | journal = Nature | volume = 459 | issue = 7245 | pages = 437–41 | date = May 2009 | pmid = 19404259 | doi = 10.1038/nature07992 | bibcode = 2009Natur.459..437S | s2cid = 4323298 }}</ref><ref name="pmid18554175">{{cite journal | vauthors = Ekker SC | title = Zinc finger-based knockout punches for zebrafish genes | journal = Zebrafish | volume = 5 | issue = 2 | pages = 121–3 | year = 2008 | pmid = 18554175 | pmc = 2849655 | doi = 10.1089/zeb.2008.9988 }}</ref><ref name="pmid19628861">{{cite journal | vauthors = Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, Vincent A, Lam S, Michalkiewicz M, Schilling R, Foeckler J, Kalloway S, Weiler H, Ménoret S, Anegon I, Davis GD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jacob HJ, Buelow R | display-authors = 6 | title = Knockout rats via embryo microinjection of zinc-finger nucleases | journal = Science | volume = 325 | issue = 5939 | pages = 433 | date = July 2009 | pmid = 19628861 | pmc = 2831805 | doi = 10.1126/science.1172447 | bibcode = 2009Sci...325..433G }}</ref> but can also be used for more standard [[gene cloning]] applications.<ref name="pmid21029755">{{cite journal | vauthors = Tovkach A, Zeevi V, Tzfira T | title = Expression, purification and characterization of cloning-grade zinc finger nuclease | journal = Journal of Biotechnology | volume = 151 | issue = 1 | pages = 1–8 | date = January 2011 | pmid = 21029755 | doi = 10.1016/j.jbiotec.2010.10.071 }}</ref> Other artificial restriction enzymes are based on the DNA binding domain of [[TAL effector]]s.<ref name="pmid20660643">{{cite journal | vauthors = Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF | display-authors = 6 | title = Targeting DNA double-strand breaks with TAL effector nucleases | journal = Genetics | volume = 186 | issue = 2 | pages = 757–61 | date = October 2010 | pmid = 20660643 | pmc = 2942870 | doi = 10.1534/genetics.110.120717 }}</ref><ref name="pmid20699274">{{cite journal | vauthors = Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B | title = TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain | journal = Nucleic Acids Research | volume = 39 | issue = 1 | pages = 359–72 | date = January 2011 | pmid = 20699274 | pmc = 3017587 | doi = 10.1093/nar/gkq704 }}</ref>

In 2013, a new technology CRISPR-Cas9, based on a prokaryotic viral defense system, was engineered for editing the genome, and it was quickly adopted in laboratories.<ref name=Hsu2014>{{cite journal | vauthors = Hsu PD, Lander ES, Zhang F | title = Development and applications of CRISPR-Cas9 for genome engineering | journal = Cell | volume = 157 | issue = 6 | pages = 1262–78 | date = June 2014 | pmid = 24906146 | pmc = 4343198 | doi = 10.1016/j.cell.2014.05.010 }}</ref> For more detail, read [[CRISPR]] (Clustered regularly interspaced short palindromic repeats).

In 2017, a group from University of Illinois reported using an [[Argonaute]] protein taken from ''[[Pyrococcus furiosus]]'' (PfAgo) along with guide DNA to edit DNA ''in vitro'' as artificial restriction enzymes.<ref>{{cite news |title=Revolutionizing Biotechnology with Artificial Restriction Enzymes |url=https://www.genengnews.com/topics/omics/revolutionizing-biotechnology-with-artificial-restriction-enzymes/ |access-date=27 May 2021 |work=Genetic Engineering and Biotechnology News |date=10 February 2017}} (reporting on [http://pubs.acs.org/doi/abs/10.1021/acssynbio.6b00324 Programmable DNA-Guided Artificial Restriction Enzymes])</ref>

Artificial ribonucleases that act as restriction enzymes for RNA have also been developed. A [[Peptide nucleic acid|PNA]]-based system, called a PNAzyme, has a Cu(II)-[[Neocuproine|2,9-dimethylphenanthroline]] group that mimics ribonucleases for specific RNA sequence and cleaves at a non-base-paired region (RNA bulge) of the targeted RNA formed when the enzyme binds the RNA. This enzyme shows selectivity by cleaving only at one site that either does not have a mismatch or is kinetically preferred out of two possible cleavage sites.<ref>{{cite journal | vauthors = Murtola M, Wenska M, Strömberg R | title = PNAzymes that are artificial RNA restriction enzymes | journal = Journal of the American Chemical Society | volume = 132 | issue = 26 | pages = 8984–90 | date = July 2010 | pmid = 20545354 | doi = 10.1021/ja1008739 }}</ref>