The classifications are not exclusive, and one enzyme can often belong several classes. In their review 51 , Nathans and Smith discuss methods for DNA cleavage and separation of the resulting fragments on gels, as well as the use of REases in other applications, e. The debate was extremely heated, but by many of the fears had abated as the anticipated dangers did not materialize and the advantages of DNA cloning, and the ability to produce large quantities of pharmaceutically important proteins such as insulin, hormones and vaccines became clear.
Type I REases were originally identified in E. They turned out to be oligomeric proteins encoded by the three h ost s pecificity d eterminant hsd genes: a restriction R , modification M and recognition S for specificity gene, respectively Table 1 and Figure 1. Before the development of DNA sequencing, genetic complementation tests defined the hsdR , hsdM and hsdS genes 53 , Other organisms will have their own families, for example, S taphylococcus aureus has at least two families [ 60 and unpublished DTFD results].
Landmark studies on purified enzymes in the wake of the Arber and Dussoix articles 17 , 61 date to They used restriction of phages fd and lambda as their assay for detecting the enzymes during purification, a laborious process 62 , The same cofactor requirement was also found for EcoBI 62 , 64 , reviewed in 25 , Twenty-five years later, we have come to appreciate that SAM, like ATP, is a widely used cofactor in many metabolic reactions 65 , A long-awaited breakthrough did not happen until much later: The structures of the subunits and assembled Type I R-M enzymes.
Two structures of S subunits appeared in and culminated in with the structure of two complete R-M enzymes containing two R subunits, two M subunits and one S subunit 67— This resulted in a heterogeneous distribution of small DNA fragments on alkaline sucrose gradients leading to the conclusion that EcoBI cuts at a variable distance from its target site. This feature is now known to be common to all Type I restriction enzymes.
This feature is also common to all Type I restriction enzymes although the distribution of cleavage locations can be broad. These were interpreted as reaction intermediates, formed by the enzymes translocating along the DNA while remaining attached to their recognition sites 72 , Recent studies confirm that on DNA binding, the enzyme strongly contracts from an open to a compact form 58 , 69 , In contrast, EcoBI appeared to form loops in only one direction.
Later studies with EcoBI did show supercoiled structures like EcoKI; however, translocation was still unidirectional, and without any apparent strand selectivity in the cleavage reaction 75 , The translocation process would explain the cleavage observed half-way between consecutive target sites on a DNA molecule as two translocating enzyme molecules would collide roughly half-way between target sites.
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. All of these steps are required to prevent indiscriminate nuclease activity The key to the functionality of the Type I REase and other SNF2 proteins is their enormous flexibility, allowing large conformational changes.
In line with such large-scale domain movement, mutational analysis of EcoR showed long-range effects, e. Type I enzymes recognize bipartite DNA sequences [e. Each variable region recognizes one part of the bipartite target sequence. A key event in understanding the significance and mechanism of variation of sequence specificity was the discovery of a brand-new specificity resulting from a genetic cross 91 , As a result of crossing-over in the conserved central region between the two variable regions, hybrid specificities were found.
This change in specificity was found to occur in vivo and in vitro and was first noted for Salmonella species 91— An extensive treatment of this topic is found in the accompanying Type I review. In Lactococcus lactis , entering plasmids may bring hsdS genes with them Site-specific recombination leads to expression of S proteins with alternative recognition domains in Mycoplasma pulmonis , thus generating combinatorial variations of recognition sequence Such plasticity of restriction specificity is also inferred in Bacteroides fragilis 97 and other species.
Type II REases are defined rather broadly as enzymes that cleave DNA at a fixed position with respect to their recognition sequence, and produce distinct DNA-fragment banding patterns during gel electrophoresis.
These REases are extremely varied and occur in many structural forms. Enzymes of this sort generally act as homodimers and cleave DNA within their recognition sequences. In vivo , they function in conjunction with a separate modification MTase that acts independently as a monomer Table 1. The first distinction made among Type II enzymes concerned REases such as HphI and FokI that recognize asymmetric sequences and cleave a short distance away, to one side.
These were designated Type IIS As the number of REases producing distinct fragments grew, it became clear that many unrelated proteins were included in the category Enzymes may exhibit more than one salient property and thus belong to more than one group. BcgI, thus, is a member of multiple groups — Type II restriction enzymes grouped by cleavage properties.
Type IIS cut away from the site e. FokI, BfiI. Type IIB require two recognition sites and cut on the outside e. Type IIE require two recognition sites, and one of the two sites acts as allosteric effector e.
Type IIF require two sites and cut at both sites as a tetramer after bringing the two regions together by looping the DNA e. See Table 2 and text for further details. Two recognition sites must be bound for activity; one is cleaved while the other acts as allosteric effector EcoRII is somewhat similar, and many REases are now known to cleave only as dimers of dimers bound to two separate sites.
Early amino acid sequences of Type II enzymes e. When crystal structures appeared — , commonalities began to emerge. This motif also appeared in other nucleases, e.
First on paper , , then via the nascent Internet by File Transfer Protocol and finally on the World Wide Web 1 , this resource makes available a focused organized data set allowing computational analysis of sequences and structures as well as access to individual topics of interest [e.
The rarity of this property 6 of surveyed suggests that any biological roles for this ability will be specialized, but the property could be used to study the ubiquitous small RNA molecules that regulate expression in all domains of life In general, Type III enzymes recognize asymmetric sequences, cleave 25—27 nucleotides away from their recognition site and use ATP and SAM as cofactors, although they do not have an absolute requirement for the latter.
Particularly interesting topics include control of the phage-borne R. EcoP1I REase activity following infection and how newly replicated DNA can be protected when only one strand of the recognition sequence is protected by methylation — An early result showed that two copies of the target site were required for DNA cleavage but that these sites had to be in a head-to-head orientation , A head-to-tail orientation prevented cleavage.
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 It appears that DNA looping may have a role in bringing the sites together , , but recent single-molecule analyses , show strong evidence for enzyme diffusion along the DNA triggered by an ATP-dependent conformational change as a novel mechanism for bringing two copies of the enzyme together to give cleavage, see also 83 , Modification-dependent restriction was first observed with populations of phage T4 that contained hydroxymethylcytosine hm5C -substituted DNA 13 , reviewed in , This original discovery relied on the fortuitous use of Shigella dysenteria SH as permissive host: it lacks both of the E.
This allowed glucoseless phage to be propagated in Shigella , while picking apart the E. Key advances in the early years lay in determining the nature of the modifications in T-even phage DNA and the genes that enable them.
The host provides the glucosyl donor , , while the phage provides the glucosyltransferase enzymes — With these genetic tools in hand, the host genes mediating the phage restriction activity were identified These were named rglA and rglB r estricts g lucose l ess phage because they mediate restriction of hmC-containing phage that lack the further glucose modification. The m5C-specific functions mcrA and mcrB were mapped and were shown to be identical to the rglA and rglB genes A third modification—dependent enzyme was found to recognize m6A as well as m5C Using the genetic tools described above, glucose-specific activity was identified , Most recently, a newly described DNA modification has provided new targets for Type IV enzymes: phosphorothioate linkages in the phosphodiester backbone.
The utility of all these discoveries was, at first, the ability to avoid them — : these restriction systems were found to underlie difficulties encountered in the introduction of foreign MTases into E. On the positive side, use of Type IV restriction in vivo also allowed enrichment of clone libraries for active eukaryotic genomic sequence, since much transcriptionally silenced DNA is heavily methylated [e.
Type IV enzymes have aroused considerable interest in recent years following the rediscovery of hm5C in the DNA of higher eukaryotes — This finding could portend the discovery of further, as yet unknown or neglected, DNA modifications. The ability of Type IV enzymes to distinguish between C, m5C, hm5C and other molecular variations of cytosine implicates these enzymes as useful tools for studies of epigenetic phenomena; the commercially available enzyme McrBC has been used for the study of such modification patterns , Much history may remain to be written.
The accompanying review focuses on structural and enzymatic properties of the systems that are known, and sketches some of the evolutionary pressures faced by restriction systems as they compete with each other and with invading replicons. Double-stranded cleavage of cellular DNA is extremely deleterious to the host cell, even when it can be repaired. Early in the study of restriction systems, the ease of moving systems among strains with differing systems by conjugation or transduction was noted.
This suggested that regulation must be present to enable exchange of activities. More recently, the sporadic distribution of R-M systems in genomes of closely related strains strongly suggests that acquisition of a new system is a relatively frequent event in nature as well.
Thus, coordination of expression or activity of the R-M activities is a key research topic. Transcriptional or translational control of Type I systems has not been documented, despite efforts to find it 8 , However post-translational control is exerted at several levels and is described in the accompanying review on Type I R-M systems.
The control of Type II R-M systems recapitulates the mechanisms for other regulatory systems and is described here. 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.
This gene is upstream of the modification gene, ecoRIM. Using primer extension to locate the start sites and gene fusions to assess expression, two adjacent promoters for ecoRIM as well as two reverse promoters were found within ecoRIR. These convergent promoters negatively affect each other [as in lambda ].
Transcription from the reverse promoter is terminated by the forward promoters and generates a small antisense RNA.
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 , The Blumenthal laboratory provided the first evidence for temporal control in the plasmid-based PvuII system of Proteus vulgaris , A similar open reading frame with similar function was also found contemporaneously in the BamHI system , Low basal expression from the pvuIIC promoter leads to accumulation of the activator, thereby boosting transcription of the C and REase genes , Figure 4.
A small C gene upstream of, and partially overlapping with, R is coexpressed from p res1 , located within the M gene, at low level with R after entry of the self-transmissible PvuII plasmid into a new host, while M is expressed at normal levels from its own two promoters p mod1 and p mod2 located within the C gene. A similar C protein operates in EspI, 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 O R , when engaged by C protein in a manner similar to that of the PvuII system.
After initial low-level expression of C. PvuII protein from the weak promoter p res1 , positive feedback by high-affinity binding of a C protein dimer to the distal O L site later stimulates expression from the second promoter p res , resulting in a leaderless transcript and more C and R protein. In this way, C protein is both an activator and negative regulator of its own transcription.
In addition, it is a negative regulator of M, which makes sense as overmethylation of DNA may also be harmful to the cell see text for further details.
The C protein binds to palindromic DNA sequences C boxes defining two sites upstream of its gene: O L , associated with activation, and O R , associated with repression. The C protein activates expression of its own gene as well as that of the REase The regulation is similar to gene control in phage lambda: differential binding affinities for the promoters in turn depend on differential DNA sequence and dual symmetry recognition.
C proteins belong to the helix-turn-helix family of transcriptional regulators that include the cI and cro repressor proteins of lambdoid phages. Two of the enzymes cut it only once, one didn't cut it at all, but Smith's enzyme cut it into eleven specific fragments. As Nathans had hoped, this broke the DNA into consistent, manageable pieces, onto which individual genetic activities could be mapped.
Using polyacrylamide gel electrophoresis, Nathans and graduate student Kathleen Danna fully separated and analyzed the structure of the SV40 fragments. They also soon determined that Smith's enzyme was actually two enzymes. They deduced the size and physical order of the fragments in the genome, and created the first cleavage maps of a viral DNA, showing where each restriction enzyme cut it. Using these maps, they were also able to identify the origin and terminus of DNA replication in the circular SV Later, they found they could actually create mutants by modifying the DNA at the known restriction sites.
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Home About Topics. Introduction Overview. Smith Courtesy Marty Katz. Smith H. A restriction enzyme from Haemophilus influenzae. Purification and general properties. Today, researchers rely on restriction enzymes to perform virtually any process that involves manipulating, analyzing, and creating new combinations of DNA sequences.
Among the many new combinations are DNA cloning, hereditary disease diagnosis, paternity testing, forensics, genomics e. Indeed, without the discovery of restriction enzymes, the fields of recombinant DNA technology, biotechnology, and genomics as we know them today would not exist. In , forty years after he purified the first restriction enzyme, Smith was part of the research team that used these very enzymes to build the first synthetic bacterial cell.
Led by Craig Venter, this team of scientists used machines to chemically synthesize the one million base-pair Mycoplasma mycoides M. Along the way, Venter and his colleagues used restriction enzymes to help clone and analyze the synthetic genome. In the final step, they transplanted the synthetic M. In this Spotlight, you'll find a broad range of resources to help you gain a deeper understanding of how restriction enzymes affected the field of molecular biology and our ability to manipulate DNA, as well as how they continue to serve as an invaluable tool for research scientists.
Watch scientists answer questions about the fundamentals of these fascinating enzymes. Read about the discovery of REs and how scientists use them. Read about how REs operate at the molecular level and how they interact with DNA at the structural level.
Learn how REs are used for hereditary disease diagnosis, paternity testing, and forensics. Watch a video about how REs helped sequence the human genome.
Learn how REs play an important role in creating genetically modified organisms. Read about how REs helped build a synthetic bacterial cell. Since , this database has organized information about REs, methylases, and the bacteria they originated from. Watch Hamilton Smith, Nobel laureate for his seminal RE research, discuss the future of synthetic genomes with a student. New England Biolabs.
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