The small size of these DNA elements probably exclude replicative forms of DNA virus, which are equal or larger than 8. Esses micoplasmas provieram de 13 rebanhos. E-mail: mironascimento hotmail. Yamamoto and A.
Elmiro R. DaMassa 2 , Richard Yamamoto 2 , M. Nascimento 3. Submitted: May 25, ; Returned to authors for corrections: August 17, ; Approved: November 12, Plasmids in Mycoplasma species class Mollicutes are unusual or rare, and only a few have been documented. Most extrachromosomal DNA isolated from mollicutes have been from viruses that are specific for mycoplasma, and the majority of the extrachromosomal DNA studies among these mycoplasma have been conducted on members of the genus Spiroplasma because of the availability of these nucleic acid elements in these organisms Since then, plasmids have been isolated from an unspeciated mycoplasma recovered from a baboon 13 , and from a caprine strain of M.
Thereafter, an unspeciated mycoplasma isolated from a goat was found to contain extrachromosomal DNA of probable plasmid origin 6. Despite the presence of some reports concerning plasmid isolations from mycoplasma, the number of isolated plasmid types is small and there is little information on the function of these DNA elements as compared to bacteria.
Additionally, information on transcriptional differences, between bacteria plasmid and the mycoplasma system 14 has incited more research in this area. In the present study, we report on the occurrence of plasmids in caprine or ovine mycoplasmal isolates caprine, 1 ovine recovered from 10 diseased and three asymptomatic herds, i. Mycoplasma strains used : One-hundred-five caprine and one ovine isolate GMA, Table 1 mycoplasma isolates were examined.
The isolates belonged do Dr. They originated from separate farms and included mycoplasma from three asymptomatic and 10 symptomatic herds showing mastitis, polyarthritis, or septicemia either singly or in combination Table 1. With the exception of M. Additionally, M. Prior to identification each mycoplasma isolate was filter-cloned a minimum of two times through nm filters according to a procedure described elsewhere For purposes of this study, the term M.
Mycoplasma strains and identification: Mycoplasmas were identified by a growth-inhibition procedure 3 modified by the use of agar wells rather than discs. Growth media, culturing, and processing : All isolates were grown for 24 to 48 hours in 50 ml of modified Hayflick liquid medium "B" described elsewhere The cultures were centrifuged for 15 minutes at 20, x G.
The cells pellets were washed twice in PBS, pH 7. DNA extraction and digestion : The DNA was extracted from mycoplasma cells by an alkaline lysis mini-preparation procedure Aliquots of the sedimented DNA from each extraction were electrophoresed in agarose gel, stained with ethidium bromide, visualized under ultraviolet light, and photographed as previously described Following digestion, samples from each reaction were electrophoresed, visualized, and photographed as described above. Mycoplasma strains: The mycoplasma examined in this study were identified either as M.
Data pertaining to the identity of the Mycoplasma , the animal species from which it was isolated, the plasmid types that were recovered, and the clinical signs caused by the mycoplasma in the herd in question are presented in Table 1.
BioCyc i. Restriction enzymes and methylases database More Search reactions for this EC number in Rhea. Short name: R. This is known as the 'taxonomic identifier' or 'taxid'. It lists the nodes as they appear top-down in the taxonomic tree, with the more general grouping listed first. ChEMBL i. DrugCentral More DrugCentral i. BindingDB database of measured binding affinities More BindingDB i.
Combined sources Automatic assertion inferred from combination of experimental and computational evidence i PDB:2E SMR i. Database of comparative protein structure models More ModBase i. PDBe-KB i. Relative evolutionary importance of amino acids within a protein sequence More The homodimer undergoes significant conformational adjustments when it assembles into oligomers , and these changes might introduce asymmetry with respect to sequence recognition.
Evolutionarily diverged versions of the same enzyme can also act in different ways. Cfr42I is a tetramer in solution, binds to two recognition sequences, and cleaves both sequences at once Eco29kI, in contrast, purifies as a monomer in solution , but binds to its recognition sequence as a homodimer, and cleaves one recognition sequence at a time ; Eco29kI also crystallizes with DNA as a homodimer ; Cfr42I has not been crystallized. Both R and M catalytic activities are harnessed to the same sequence-specificity module S , which can occur as a separate subunit or as the C-terminus of the RM protein.
S-modules can recognize single continuous DNA sequences, or bipartite discontinuous sequences, either of which can be symmetric or asymmetric. Type IIG enzymes occur in a variety of oligomeric forms, with or without separate, accompanying MTases.
AdoMet is the donor of the methyl group, and so it is essential for the methylation reaction. Since it also either stimulates, or is absolutely required for, the cleavage reaction, it likely acts as an allosteric activator, too. The advantage of AdoMet dependency again might be self-preservation, since it reduces the likelihood that the cell's own DNA will be cleaved at times of AdoMet shortage and consequent undeR—Modification.
A proportion of sequences become modified during incubation, and thereafter are resistant to cleavage The enzyme comprises an N-terminal endonuclease domain, a central gamma-class methyltransferase domain, and a C-terminal specificity domain. Structural comparisons and modeling show that in order to bind DNA specifically, the C-terminal domain of BpuSI must rotate with respect to the R and M domains, and reorganize.
If the DNA sequence recognized by Type IIG enzymes changes—by mutations in the S-module , for example, or by domain exchange —it does so for both restriction and modification activities in the same way at the same time. This functional synchrony has allowed the specificities of certain Type IIG enzymes, such as those of the MmeI-family, to diverge widely in the course of evolution.
Numerous MmeI-family enzymes have been characterized, each similarly organized and similar in aa sequence and hence structure, but specific for a different 6—8 bp recognition sequence and Supplementary Table S1, group E.
The C-alpha backbone of the recognition domain of these proteins has evolved a conformation that allows different pairs of amino acids to specify alternative base pairs in the sequence recognized. Other pairs of amino acids within the specificity domain determine other base pairs in the recognition sequences. This is unusual behavior for restriction enzymes, which as a whole have evolved in the other direction, toward recognition sequence immutability, instead When such hemimethylated sequences replicate, one daughter duplex retains the hemimethylation, but the other becomes completely unmethylated.
How unmethylated daughter sequences are distinguished from foreign DNA is unclear, but it seems likely that pairs of sequences in opposite orientations, and perhaps several pairs, are monitored before the enzyme commits to either cleavage or re-methylation.
MmeI-family enzymes cleave substrates with multiple sites more efficiently than substrates with single sites, and cleavage is stimulated by the addition of oligos that contain a recognition site. The enzymes purify as monomers, but there are strong indications that they cleave as homodimers or higher-order oligomers formed between enzyme molecules bound to adjacent, opposed recognition sites.
When modeled, the structures of these complexes closely resemble Type I REases, with the difference that Type IIG cleavage domains cut DNA at fixed positions close to their recognition site s whereas Type I R-subunits cleave at variable distances, far away. Unless we misread the situation, enzymes of this kind perform some interesting gymnastics in the course of their cleavage reactions , The catalytic complexes of Type IIG enzymes are likely to be large and difficult to solve by crystallography.
Alternative approaches such as single particle cryo-electron microscopy and reconstruction , , , or molecular modeling , might prove fruitful in the interim. Extreme differences can be found, however. Borrelia burgdorferi isolates, in contrast, can have up to 20 Type IIG systems, to the complete exclusion of all other types.
We know little about the selective advantages and disadvantages that underlie these variations. The M. AhdI is one, are widespread and adaptable, and accompany many Type II REases, both those recognizing continuous sequences e. The Type IIH distinction seems less important, now, as also do several of the other Type II sub-classifications, and it is rarely used.
Type IIM enzymes require m ethylated recognition sequences. Both domains bind DNA in a sequence- and methylation-dependent manner. DpnI has been crystallized with DNA bound at the C-terminal effector domain, but not at the catalytic domain The complementary specificities of DpnI and DpnII proved to be very useful for site-directed mutagenesis experiments.
It remains unclear what structural features of DpnI account for its absolute dependence on methylated adenines within its recognition sequence. Methyl groups can increase the affinity between a protein and a DNA sequence through hydrophobic interactions, but this will hardly produce the all-or-nothing behavior seen among the methylation-dependent REases.
It is possible, instead, that the methyl groups induce a structural change by, for example, altering the side-chain conformations of long-chain amino acids such as arginine and lysine, switching them from conformations that interfere with and prevent binding, to conformations that are compatible with and permit binding.
They recognize symmetric p alindromic sequences and cleave symmetrically within the sequence e. Some Type IIP REases act as monomers, but most act as homodimers or homotetramers, and this structural duplication accounts for their symmetry in specificity and catalysis.
The multimers generally, but not always , cleave both strands of the DNA duplex in the same binding event. The monomers cleave DNA one strand at a time, but without the release of nicked intermediate, indicating that the same enzyme molecule cleaves both DNA strands at each recognition sequence , first one strand and then the other. This they do without detaching from the DNA and returning to bulk solution Type IIP recognition sequences are usually 4—8 specific base pairs in length.
They can be continuous e. Recognition sequences can comprise a single base pair e. Hundreds of different Type IIP specificities are known. For each, usually several, and sometimes very many, REases of identical specificity and similar amino acid sequence can be found in other bacteria and archaea. Even among related enzymes, significant differences in biochemical behavior have been noted REases with unrelated specificities generally display no amino acid sequence similarity, however, signifying either that no trace of common ancestry remains due to the passage of time, or that they arose independently to begin with.
Cleavage is s hifted to one side of the sequence, within one or two turns of the double helix away. Type IIS enzymes were first discerned as being different by Waclaw Szybalski and colleagues at the University of Wisconsin , who devised a variety of ingenious applications for them , FokI, one of the earliest such enzymes discovered , is the best known and is the source of the DNA-cleavage domain used in synthetic gene-targeting endonucleases These form a close-knit group centered on their core gamma-class MTase domain, as described above.
They are distinct from the rest of the Type IIS enzymes, and are excluded from the discussion that follows. FokI has been studied in some depth, and has been crystallized with and without bound DNA , — In Type IIP REases, the amino acids responsible for recognition and for catalysis are integrated into one composite domain Type IIS recognition sequences are usually asymmetric.
In all likelihood this is not through necessity, but rather reflects the fact that far more asymmetric DNA sequences exist to be recognized than symmetric sequences. Because the recognition sequence is asymmetric, cleavage takes place on only one side.
If it were symmetric, both sides would become cleaved, first one and then the other. Type IIS REases are usually accompanied by two separate MTases, each of which modifies one strand of the recognition sequence by methylating one adenine or one cytosine in that strand. Often, these MTases occur as individual proteins, but sometimes, as is the case in the FokI R—M system, they are joined into one protein chain , The benefits of such fusions are unknown but, all things being equal, it allows the MTases to be synthesized in a fixed, ratio and their synthesis to be co-regulated.
And, if the hemimethylated daughter DNA duplexes are re-methylated as they emerge from the replication complex, whenever one MTase is needed to service one duplex, the other MTase is on hand to service the other duplex.
These systems recognize quasi-palindromic sequences that are viewed as a symmetric by the REase, but symmetric and ambiguous by the MTase. As a consequence, both strands of the recognition sequence become modified by just the one MTase. There is a price to be paid for methylation, and prokaryotes go to lengths not to squander it. A single MTase such as M. MboI, M. In the crystal structure of SfiI with DNA, the catalytic sites are too far from the DNA to initiate cleavage, exemplifying perhaps another cleavage-control mechanism Second, nicked DNA intermediate does not accumulate during the FokI cleavage reaction, suggesting that an individual cleavage domain cannot catalyze strand-cleavage on its own.
And third, cleavage is stimulated by multiple recognition sites in the DNA, and by the addition of the purified catalytic domain, suggesting that cleavage of duplex DNA requires the dimerization of two catalytic domains Pieces of the cleavage puzzle are still missing and await further experimentation, but the current idea is that double-strand cleavage by Type IIS REases requires dimerization of the catalytic domains of nearby molecules at least one of which is specifically bound to a recognition site , In some cases the second molecule can be free in solution or bound to DNA non-specifically , but the complex is more stable when it, too, is specifically bound , If so, the two sites do not have to be nearby, or in any particular orientation, and if they are far apart, DNA looping takes place between them , A surprisingly large number of Type II REases behave in this way, in fact, and require at least two recognition sites in order to cleave — This is somewhat surprising because in order to dimerize, the catalytic domains must assume opposite orientations.
It is easier to visualize this happening between molecules that approach one another head-on, than sideways. Ultimately in the synapses, both bound DNA duplexes become cleaved. This suggests that the catalytic domains can shift from one side to the other, cleaving first one duplex and then moving over to cleave the other.
The co-crystal structure of FokI reveals how the DNA interacts specifically with the binding domain pdb:1FOK , but not how it interacts with the cleavage domain s In fact, for all of the many REases we suppose dimerize transiently through their catalytic domains when they cleave DNA, not a single structure revealing this event has been obtained.
Instead we rely on modeling. Using a clever combination of two FokI mutant enzymes, one DA binding-proficient but catalysis-deficient, the other N13Y binding-deficient but catalysis-proficient, Steve Halford's group confirmed this strand-specificity experimentally. Whether the same strand-specificity holds true for other Type IIS enzymes remains to be seen.
Type IIT enzymes, today, are perhaps more usefully defined as REases that have t wo different catalytic sites. Some of these enzymes are heterodimers e. Others are single-chain proteins with two distinct catalytic domains e. All of these enzymes recognize asymmetric sequences and cleave within, or very close to, only one side of the sequence. In some systems, these MTases are individual proteins, in others they are joined into a single protein chain. Because Type IIT REases, as defined here, have two different catalytic sites they can be converted into strand-specific nicking endonucleases by mutating one site or the other , , or by eliminating the small subunit See Chan et al.
Eco29kI for the amino acid motifs that comprise their catalytic sites. These motifs recur in other kinds of nucleases, including homing endonucleases, Holliday-junction resolvases and exonucleases 8.
Non-specific endonucleases e. Sokolowska et al. DpnI and variants e. Vsr And unlike conventional zinc-finger domains that function in DNA sequence recognition, those of HNH REases—there can be more than one in each subunit—perform structural roles unrelated to sequence recognition. This is same coordination as occurs in the HNH REases discussed above, except that an extra water molecule takes the place of the second amino acid, Asn. Eco29kI crystallized without a metal ion at the catalytic site but the organization is similar.
In both structures, the nucleophilic water molecule is positioned and oriented to attack the phosphorus by H-bonds to one main chain carbonyl oxygen and to the side chain oxygen of the tyrosine of the first GIY motif. In this position, the metal ion is beyond coordination range of the nucleophilic water, which cannot therefore originate from its hydration sphere.
The invariant metal ion of PD-EXK sites, on the other hand, contacts only the non-bridging phosphate oxygen, and is often close enough to coordinate, and help orient, the nucleophilic water. They use a metal-independent catalytic site, termed PLD belonging to the Phospholipase D superfamily, and they cleave DNA one strand at a time in an unusual way involving a covalent enzyme—DNA intermediate BfiI acts as a homodimer.
The C-terminal half of each subunit forms a DNA-binding domain, which resembles B3-like plant transcription factors , The dimer binds to two recognition sequences at once but has only one catalytic site, which is located at the interface of the two N-terminal domains, as it is in the EDTA-resistant Nuc endonuclease from Salmonella typhimurium The same catalytic site then transfers to the top DNA strand to hydrolyze that.
In principle, since the catalytic site of BfiI is symmetric, it should be able to accommodate the opposite polarity of the top strand by switching the roles of the two histidines and working in reverse, as was originally proposed It is easy to imagine these enzymes binding to their quasi-symmetric recognition sequence as homodimers with a single composite catalytic site.
It will be interesting to see whether this catalytic site is bi-directional and can work in reverse, or if these enzymes detach, rotate and reattach in order to hydrolyze both strands, much like the monomeric Type IIP REases such as BcnI Its genomic location suggested it might mediate genetic rearrangements, and its proximity to the gene for a companion MTase implied that it was a small, unremarkable, Type IIP REase.
The crystal structure of PabI with specific DNA was solved very recently, and shows why this enzyme is so unusual: it is not an endonuclease, after all PabI binds to its symmetric recognition sequence as a homodimer, and flips all four purines out from the helix, leaving the pyrimidines intra-helical, but orphans.
It leaves the phophodiester backbone intact, and instead excises both adenine residues to create apurinic sites opposite the thymines.
At the high temperature at which P. Close isoschizomers of PabI are ubiquitous in strains of H. More can be assigned to the PLD-family or the PabI-group, but others cannot be assigned to any family and remain mysteries 26 , They could be fringe members of the conventional families that have diverged beyond recognition or, like PabI, they could be examples of new, as yet uncharacterized, folds and DNA-degradation mechanisms. Type II REase quarternary organizations can often be understood in terms of the different ways in which two catalytic sites can be brought to act on opposite strands in the vicinity of the same DNA sequence.
Surprisingly, they do this without detaching from the DNA and returning to bulk solution. Instead, they release the recognition sequence after the first nick, and then randomly slide along the DNA and rotate until the sequence is recaptured in opposite orientation Other monomers, such as BsrI and MvaI , represent single-chain fusions of ancestral heterodimers.
They possess two different catalytic sites within the one polypeptide chain, and generally cleave both DNA strands in one binding event. These enzymes can exhibit marked strand preference, such that one strand must be cleaved by one of the catalytic sites before the second can be cleaved by the other catalytic site Whether this is due to a structural peculiarity of the second catalytic site, or to a biochemical peculiarity in the way it catalyzes the reaction, is not known.
Activation is thought to occur by transient dimerization of the catalytic domain with an identical catalytic domain from a second enzyme molecule either bound to another recognition site or, with lesser effect, unbound.
Dimerization activates both catalytic sites, and so these enzymes generally cleave both DNA strands at once without the release of nicked intermediates. The need for transient dimerization accounts for the low activity of many REases on substrates with only one recognition site, and explains why activity often increases in the presence of oligos that contain the recognition sequence.
Many restriction enzymes cleave DNA as multimers bound to two recognition sequences at once. Such widespread behavior must confer a significant selective advantage, one that has to do, perhaps, with carefully distinguishing host DNA that must be saved, from foreign DNA that must be destroyed.
REases that cleave by transient dimerization automatically monitor two sequences at once when both members of the partnership are bound to DNA specifically. This might be the underlying reason why so many REases operate in this way instead of simply acquiring a second catalytic site, a trivial step in evolutionary terms.
REases that bind to their recognition sequences as homodimers already have both catalytic sites needed for cleavage. Some, such as NgoMIV and SfiI, nevertheless monitor two recognition sequences at once by assembling into tetramers of two back-to-back homodimers. Soon after the structure of the EcoRI—DNA complex was determined , attempts were made to change the specificity of EcoRI by substituting the amino acids involved in base-specific interactions for example Substitutions were made according to suggestions a decade earlier that certain amino acids were ideally suited to juxtapose certain bases due to H-bond complementarity.
Asparagine and glutamine were ideal for adenine, it was proposed, and arginine was ideal for guanine The substitutions introduced into EcoRI and EcoRV, and subsequently into other REases, usually resulted in a decrease in activity, but without exception failed to produce substantial changes in specificity.
The reason for these failures has become clearer with time: recognition is a highly cooperative and redundant process, involving not only amino acids in direct contact with the bases and the backbone, but also structured water molecules and an intricate network of buttressing interactions Even for very well characterized REases, the properties that determine specificity and selectivity are difficult to model with the available structural information In order to change specificity, the functional groups of amino acids must be positioned in three dimensions within the DNA-binding site in precise complementarity with the bases they are to juxtapose.
This demands structural accuracy far beyond what can be achieved by gross amino acid substitutions. Notwithstanding, some Type IIG combined RM enzymes have evolved DNA-binding domains with C-alpha structures that allow them to undergo specificity changes naturally at certain base pair positions.
Such changes confer a selective advantage because it allows prokaryotes to side-step the resistance to restriction that constantly evolves among its viruses. Almost invariably, these changes in specificity involve switches of two amino acids at once—one for each base of the base pair—and they can be replicated in the laboratory by site-specific mutagenesis to achieve robust changes of specificity.
For example, in the MmeI-family of highly homologous RM enzymes that recognize 6—8 bp asymmetric sequences, specificity for GC at certain positions can be routinely changed to CG, and vice versa, by substituting Glu…Arg E…R pairs for Lys…Asp K…D pairs, and certain other equivalent amino acid combinations Nicking enzymes can be isolated as the principal large subunits of some heterodimeric REases , , — , or they can be engineered by generating homodimers — or heterodimers with one active catalytic site and one inactive catalytic site The former enzymes are unusual because their catalytic sites can act alone.
One aa recognizes the DNA and contains the catalytic site for top-strand hydrolysis; the other aa contains the catalytic site for variable hydrolysis of the bottom strand.
Type II REases are among the most specific enzymes known. For precise gene targeting in the complex genomes of eukaryotes, only a single cut at a defined location is desirable.
Achieving this degree of specificity requires a recognition sequence of about 20 bp in length. Whereas the catalytic and recognition residues of the latter are integrated into a single protein domain, in FokI they are separate. ZFNs typically contain a series of three to six zinc fingers. Mode of DNA binding by zinc finger proteins: each finger recognizes approximately three base pairs of the recognition sequence.
For one zinc finger the amino acids forming essential base contacts residues at positions 1, 2, 3, 6 of each helix are shown in purple.
The specificities of individual fingers can be changed to some extent by mutagenesis, and the order of the fingers in an array can be changed at will by gene synthesis. In principle, almost any sequence in a complex genome can be targeted with a carefully selected zinc finger array, although in practice this is easier said than done.
The non-specific FokI cleavage domain of ZFNs does not contribute to specificity, but it has a property that greatly enhances the accuracy and utility of ZFNs. On its own, the FokI CD is inactive. Pairs of ZFNs have been used with considerable success in this way for gene targeting — , although evidence is mounting that they are not as specific as might be expected , , and that cleavage at unintended sites also occurs.
Part might also be due, as pointed out by Halford et al. The FokI CD is inherently compromised, they suggest, because its dimerization mechanism does not preclude off-site targeting Recently, a novel zinc-finger nuclease platform was described using a derivative of PvuII as a sequence-specific catalytic domain instead of the FokI CD. The design and selection of zinc finger arrays to make pairs of ZFNs for gene targeting is complex and costly.
After the DNA-binding domains of transcription activator-like effector TALE proteins were shown to be modular, and to recognize DNA in a simple 1 module:1 base fashion , , they began to be used instead of ZF arrays to engineer programmable nucleases for gene targeting. The repeat arrays form a right-handed super-helix that spirals around the DNA with astonishing elegance, following the track of the major groove for several turns.
The repeats precisely track the sense strand of the DNA, and so the order of the repeats determines the bp sequence recognized. Gene targeting requires precisely positioned incisions in genomic DNA in order to stimulate repair by homology-directed genetic recombination HR. It has been argued that it might be better to cut only one DNA strand for this purpose, using a nicking domain rather than a cleavage domain, as this would decrease competing repair by error-prone, non-homologous end joining NHEJ Such engineered nickases have been used in conjunction with zinc fingers — , TALE proteins and methyl CpG binding domains , and are proving to be very effective.
Type II REases have come of age. In doing so, they have changed the landscape of molecular biology in ways barely imaginable a few decades ago. It all started with the observation that phage sometimes infect new bacteria very poorly. This technology has since transformed the life sciences and medicine, and has seeded a multitude of enterprises, large and small To Type II REases we owe many billions of dollars of economic activity, thousands of jobs and careers, and staggering advances in knowledge and understanding.
Few examples as this speak so clearly of the importance to society of investments in unencumbered, curiosity-driven, basic research.
We thank our colleagues for their many contributions to the field of restriction and modification, particularly those whose work we have not cited due to lack of space.
We thank the anonymous reviewers of early versions of this article whose comments and suggestions improved it greatly. Conflict of interest statement. The authors declare no conflicting financial interest.
National Center for Biotechnology Information , U. Journal List Nucleic Acids Res v. Nucleic Acids Res. Published online Jun Geoffrey G. Wilson 2 New England Biolabs Inc. Author information Article notes Copyright and License information Disclaimer. This article has been corrected. See Nucleic Acids Res. This article has been cited by other articles in PMC. Open in a separate window. Figure 1.
Figure 2. The structural basis of specificity of REases: characterization of the REase—DNA interface using modified substrates Because Type II REases recognize their substrate sequences so accurately, they are attractive subjects for studying the mechanism of recognition.
Figure 3. Thermodynamics and kinetics of DNA binding The affinity of a REase for its substrate sequence was determined for EcoRI using the nitrocellulose filter-binding technique that had been developed in the mids , Facilitated diffusion, linear diffusion, sliding and hopping Detailed investigation of the kinetics of the EcoRI-substrate interaction revealed a surprising result 10 , Large-scale purification of REases from overproducing E.
Figure 4. Figure 5. EcoRV The structure of EcoRV, the next to be crystallized after EcoRI, was solved in multiple forms, including the free enzyme apo-protein , specific enzyme—DNA complexes, an enzyme—product complex and, revealingly, a non-specific complex , Figure 6. The mechanism of catalysis One of the most important questions regarding the catalytic mechanism of a hydrolase is whether hydrolysis involves a covalent intermediate, as is typical for proteases.
Figure 7. Figure 8. Figure 9. Figure Grouping by quarternary structure Since the substrates of REases are duplex DNA molecules, cleavage requires two catalytic reactions, one for hydrolyzing each DNA strand. Protein engineering of REases—tools for gene targeting REase variants Soon after the structure of the EcoRI—DNA complex was determined , attempts were made to change the specificity of EcoRI by substituting the amino acids involved in base-specific interactions for example Acknowledgments A.
Loenen W. Type I restriction enzymes and their relatives. Rao D. Type III restriction-modification enzymes: a historical perspective. The other face of restriction: modification-dependent enzymes.
Highlights of the DNA cutters: a short history of the restriction enzymes. Mruk I. To be or not to be: regulation of restriction—modification systems and other toxin—antitoxin systems. Roberts R. How restriction enzymes became the workhorses of molecular biology.
Pingoud A. Type II restriction endonucleases: structure and mechanism. Life Sci. Restriction Endonucleases. Berlin, Heidelberg, New York: Springer; Modrich P. In: Nucleases. Smith H. A suggested nomenclature for bacterial host modification and restriction systems and their enzymes. Restriction endonucleases. CRC Crit. A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes.
Van Etten J. Chlorella viruses code for restriction and modification enzymes. Zhang Y. Nelson M. Chlorella viruses encode multiple DNA methyltransferases.
Schumann J. Noyer-Weidner M. Behrens B. Organization of multispecific DNA methyltransferases encoded by temperate Bacillus subtilis phages. EMBO J. Gasiunas G. Shen B. This paper describes a novel method for differentiating C. The 16S rRNA gene sequences for all known lipophilic Corynebacterium species were obtained from published data and analyzed.
The method was successfully employed to identify of
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