discovery of restriction enzymes
See Table 2 for the complexity and diversity of Type II subtypes. In 1965 Arber argued that should it be possible to isolate and characterise restriction enzymes, they could be used as a laboratory tool to cleave DNA. Tel: +1 978 380 7370; Fax: REBASEa database for DNA restriction and modification: enzymes, genes and genomes, Structure and substrate recognition of the Escherichia coli DNA adenine methyltransferase, Cytosines do it, thymines do it, even pseudouridines do itbase flipping by an enzyme that acts on RNA, Surviving the sun: repair and bypass of DNA UV lesions, Recent advances in the structural mechanisms of DNA glycosylases, DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O6-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy, Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle), 2001 Fred Griffith review lecture. Credit: David Gregory and Debbie Marshall, Wellcome Images. The technical ingenuity applied to the use of restriction enzymes warrants a separate detailed Survey and Summary or indeed an entire book. This feature is also common to all Type I restriction enzymes although the distribution of cleavage locations can be broad. EcoRI, EcoRV, PvuII and BamHI) showed them to be almost completely unrelated (105109). Enzymes such as BcnI act as a monomer, in contrast to most Type II REases that act as dimers. Such strains pose a great threat to humans and animals (270). The control of Type II R-M systems recapitulates the mechanisms for other regulatory systems and is described here.
One cut up the DNA of the virus and the other restricted its growth. Attendees of the 1972 EMBO Workshop helped identify the people in Figure 2. Identification of allergens and allergen hydrolysates by proteomics and metabolomics: A comparative study of natural and enzymolytic bee pollen. Amplification and preparation of the gene product, T4 polynucleotide kinase; cloning of the gene (pseT) and amplification of its product, pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long, Phage lambda receptor chromosomes for DNA fragments made with restriction endonuclease I of Bacillus amyloliquefaciens H, A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes, Novel bacteriophage lambda cloning vector, The isolation of structural genes from libraries of eucaryotic DNA, Rec-mediated recombinational hot spot activity in bacteriophage lambda. The EcoRI REase was found to cleave GAATTC (39,40) and the corresponding M.EcoRI MTase to modify the inner adenines in this sequence, producing GAm6ATTC (41). 
Gunther Stent suggested that DNA methylation might be the basis for the modification imprint, thus prompting Arber to show that methionine was required in the growth medium to produce the imprint on the DNA (27).
The modification-dependent Type IV enzymes are highly diverse and only a few have been characterized in any detail. This important finding coincided with the discovery of RNA-methyltransferase and MTase activities in bacteria that catalysed the formation of m5C and m6A (28). The first of these new enzymes, HindII, was discovered in Hamilton (Ham) Smiths laboratory at Johns Hopkins Medical School in 1970 (29). A similar C protein operates in Esp1396I, but in this case the genes are convergently transcribed with transcription terminator structures in between, and M is expressed from a promoter under negative control of operator OR, when engaged by C protein in a manner similar to that of the PvuII system. They are produced as part of an effort to generate restriction-modification enzymes with longer recognition sites without having to screen bacteria and microorganisms. Different specificities could be imprinted concomitantly both by the bacterium itself (by what were later recognized to be the Type I EcoKI and EcoBI systems) and by phage P1 in its latent prophage state (the Type III EcoP1I system). The S subunit has a duplicated organization: two 150 aa variable regions alternate with smaller conserved regions, which are highly similar within each of the five families. Here, PhD student Robert Yoshimori (37) benefited from the experience of Daisy Dussoix, who had moved from Werner Arbers lab to UCSF. Such bacteria are an important source for restriction enzymes. AAC(N6)GTGC for EcoKI]. In 1995 he and a team at the Institute for Genomic Research completed the DNA sequence of Haemophilus influenzae. On the species specificity of the methylation enzymes, Digestion of deoxyribonucleic acids from bacteriophage T7, lambda, and phi 80h with site-specific nucleases from, Recognition sequence of restriction endonuclease III from. None declared. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine.
Far fewer Type I, III and IV restriction enzymes have been characterised, but studies of these enzymes are providing rich information on DNAprotein interactions and catalysis, protein family relationships, control of restriction activity and plasticity of protein domains. This feature is now known to be common to all Type I restriction enzymes. These findings were presented at the 1972 EMBO Workshop on Restriction, organized by Werner Arber (see Supplement S1 for details of the program and attendees). The new restriction enzymes are called Zinc finger nucleases (originally called chimeric restriction enzymes). Intricate control of restriction in the operons of the Type II R-M systems of PvuII and Esp1396I by controlling C proteins. The host provides the glucosyl donor (151,152), while the phage provides the glucosyltransferase enzymes (153155). If this last idea should be correct one may further speculate that a restriction enzyme might provide a tool for the sequence-specific cleavage of DNA (22) (our double quotes). Subsequently, what was thought to be pure HindII was found to be a mixture of HindII and a second REase made by the same bacterium, HindIII. These were designated Type IIS (98). In addition, vectors carrying the intact gene but with multiple cloning sites allowed EcoRI-based DNA constructs for transcriptional and translational fusions to the lacZ gene (238250). Entry of a second R-M system thus becomes lethal, a phenomenon called apoptotic mutual exclusion (211). BcgI, in contrast, is complicated since it recognizes an asymmetric DNA sequence (= Type IIA); cleaves on both sides of that sequence (= Type IIB); and comprises a fused endonuclease-methyltransferase subunit (= Type IIC) plus a Type I-like DNA-specificity subunit (= Type IIH). Type IV enzymes have aroused considerable interest in recent years following the rediscovery of hm5C in the DNA of higher eukaryotes (172175). HindIII cleaved DNA at a different symmetric sequence, AAGCTT (31,32); [see (33) for a thought-provoking discussion]. In each case, an example is given of one of the best-characterized enzymes within the different Types I, II and III. Double-stranded cleavage of cellular DNA is extremely deleterious to the host cell, even when it can be repaired.
His work inspired the use of restriction enzymes for many different biotechnology applications, including DNA sequencing and the construction of recombinant DNA. An end to 40 years of mistakes in DNA-protein association kinetics? Boxes outline functions that are filled by distinct protein domains. Photograph of the participants at the EMBO Workshop on restriction in Leuenberg (Basel), Switzerland, 2630 September 1972, organized by Werner Arber, who took the picture (Archive Noreen Murray). Eop values would range from 101 to 105, thus indicating that R-M systems were effective barriers to the uptake of DNA; see (16,2226) for early reviews. The Type II restriction enzymes (e.g. This finding could portend the discovery of further, as yet unknown or neglected, DNA modifications. Other organisms will have their own families, for example, Staphylococcus aureus has at least two families [(60) and unpublished DTFD results]. He was awarded the Nobel Prize in Physiology or Medicine in 1978 for his work on restriction enzymes. Two recognition sites must be bound for activity; one is cleaved while the other acts as allosteric effector (104). Currently, Rebase lists 19 documented C proteins, as well as 432 putatives based on sequence data (16 September 2013, http://rebase.neb.com). As such, Type I enzymes could prove useful for understanding the action of SNF2 enzymes in higher organisms, including the coordinated steps of DNA scanning, recognition, binding and alteration of the helical structure, that allow other domains or subunits to move and touch the DNA. Enabling technical advances included development of electron microscopy, ultracentrifugation, chromatography, electrophoresis and radiographic crystallography.
(1971) A genetic and biochemical analysis of the restriction and modification of DNA by resistance transfer factors. Modification-dependent restriction was first observed with populations of phage T4 that contained hydroxymethylcytosine (hm5C)-substituted DNA (13), reviewed in (149,150). This activity may seem of arcane interest, but a broader understanding especially of the translocating enzymes could further understanding of genome stabilization activities in all domains of life. The C protein activates expression of its own gene as well as that of the REase (188). How this communication between the two target sites was achieved when ATP hydrolysis was insufficient for DNA translocation like the Type I enzymes (59) has provoked much discussion (141). The structures were solved without bound DNA, and while they confirmed the close relationship between C-proteins and helix-turn-helix DNA-binding proteins in general, they did not reveal details of the interactions between C-proteins and their C-box binding sites in DNA (195,197204). S-adenosylmethionine: jack of all trades and master of everything? Type I and II are currently divided in 5 and 11 different subclasses, respectively. FokI, BfiI). When crystal structures appeared (110114), commonalities began to emerge. Today restriction enzymes are an indispensable tool for biotechnology. BcgI, thus, is a member of multiple groups (100102). Low basal expression from the pvuIIC promoter leads to accumulation of the activator, thereby boosting transcription of the C and REase genes (108,185) (Figure 4). The power of restriction enzymes to cut DNA was demonstrated by Kathleen Danna, a graduate student, with Daniel Nathans, her doctoral supervisor, at Johns Hopkins University. II. As Smiths work proceeded on the east coast of the USA, REases with similar behaviour but different specificity were discovered in the laboratory of Herb Boyer at the University of California, San Francisco, on the west coast.
The presence of the antisense RNA gene in trans reduced lethality mediated by cleavage of under-methylated chromosomes after loss of the EcoRI plasmid (post-segregational killing) (182,184). I. Purification and general properties',Journal of Molecular Biology, 51/2 (1970), 379-91. Yoshimori investigated restriction systems present on plasmids in clinical E. coli isolates, and purified what became known as EcoRI and EcoRII (37,38). The debate on the safety of recombinant DNA technology started soon after the 1972 EMBO Workshop and reports on the transfer of eukaryotic DNA into E. coli [documented by (52)]. Cancer immunotherapy | CRISPR-Cas9 | DNA | DNA extraction | DNA polymerase | DNA Sequencing | Epigenetics | Faecal microbiota transplant | Gene therapy | Immune checkpoint inhibitors | Infectious diseases | Messenger RNA (mRNA) | Monoclonal antibodies | Nanopore sequencing | Organ-on-a-chip | p53 Gene | PCR | Phage display | Phage therapy | Plasmid | Recombinant DNA | Stem cells | The human microbiome | Transgenic animals |. Before the development of DNA sequencing, genetic complementation tests defined the hsdR, hsdM and hsdS genes (53,54). Note that Type II enzymes range from simple (shown here for EcoRI) to more complex systems (see Table 2 for the diversity of Type II subtypes). Sau3AI (GATC) is a monomeric Type IIE REase that dimerizes on the DNA, inducing DNA loops. Each variable region recognizes one part of the bipartite target sequence. In contrast, EcoBI appeared to form loops in only one direction. Arber believed these two enzymes could provide an important tool for cutting and pasting DNA, the method now used in genetic engineering. These reports of host-controlled variation in bacterial viruses were reviewed by Luria (1953) (16).
This made possible rapid screening of bacterial colonies on an agar plate for those lacking the activity of LacZ using a colour assay. More recently they have been redesigned to create artifical nucleases, the Zinc-finger nucleases and the TAL-effector nucleases, which have potential for gene targeting and gene therapy (212). For example, the actions of translocating enzymes such as the Type I and IV enzymes at a replication fork or other variant structure are one such possibility (272,273). Sequencing and biochemistry have since led to subdivisions within the Type I (see below) and Type II systems (Table 2) [see (49,50) and http://rebase.neb.com for nomenclature and details]. They published their findings in 'Host specificity of DNA produced by Escherichia coli I and II', Journal Molecular Biology, 5 (1962), 1836 and 37-49. Soon after, in July 1970, Hamilton Smith and Kent Wilcox announced that they had isolated and characterised a restriction enzyme (HindII) in a second bacterial species, Haemophilus influenza, and demonstrated that it degraded the DNA of a foreign phage. Most of the Type II systems that have been examined have the problem of integrating control of the modification and restriction activities separately, since they are embodied in separate proteins. For instance, their use led to the production of insulin from recombinant bacteria and yeast by Genentech, thus greatly increasing the supply for diabetics and the production of a recombinant vaccine for Hepatitis B by Biogen to treat the hundreds of millions of people at risk of infection by this virus. Type I enzymes recognize bipartite DNA sequences [e.g. These were named rglA and rglB (restricts glucoseless phage) because they mediate restriction of hmC-containing phage that lack the further glucose modification. Fittingly, in 1978, Werner Arber was awarded the Nobel Prize together with Dan Nathans and Ham Smith in recognition for their pioneering work on R-M (www.nobelprize.org). Restriction endonucleases (REases) such as EcoRI are familiar to virtually everyone who has worked with DNA. Arber indicated in 1965 that restriction enzymes could be used as a tool for cleaving DNA. Cleavage of a model DNA replication fork by a Type I restriction endonuclease, Cleavage of a model DNA replication fork by a methyl-specific endonuclease, DNA repair endonuclease ERCC1-XPF as a novel therapeutic target to overcome chemoresistance in cancer therapy. Few enzymes have been well-characterized, but based on the current avalanche of sequence information many putative genes belonging to all Types and subtypes are being identified and listed on the restriction enzyme website (http://rebase.neb.com).
Since the recognition sites are symmetric, this means that every fragment is flanked by the same single-stranded extension, allowing any fragment to anneal (via the extensions) to any other fragment, thus setting the stage for recombining DNA fragments and cloning. The use of REases in this system enabled the creation of suitable procedures for such identification, although PCR has largely displaced REases in this application. Smith was awarded the Nobel Prize for Physiology or Medicine in 1978 for his part in the discovery of the enzyme. DpnI (Gm6ATC) is a Type IIM REase, which cleaves its recognition sequence only when the sequence is methylated (103). Preliminary descriptions of the phenomenon of R-M were published by Luria and Human (1952) (13), Anderson and Felix (1952) (14) and Bertani and Weigle (1953) (15).
They cut 3H-labelled double-strand RF DNA of phage f1 (a relative of phage M13) with EcoBI, denatured and renatured the DNA and then treated with EcoBI a second time.
