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Background

We previously defined a family of restriction endonucleases (REases) from Thermus sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (MTase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by S-adenosylmethionine (SAM), and incomplete cleavage of the substrate DNA. Members include related thermophilic REases with five distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI.

Results

TspDTI, TsoI and isoschizomers Tth111II/TthHB27I recognize different, but related sequences: 5'-ATGAA-3', 5'-TARCCA-3' and 5'-CAARCA-3' respectively. Their amino acid sequences are similar, which is unusual among REases of different specificity. To gain insight into this group of REases, TspDTI, the prototype member of the Thermus sp. enzyme family, was cloned and characterized using a recently developed method for partially cleaving REases.

Conclusions

TspDTI, TsoI and isoschizomers Tth111II/TthHB27I are closely related bifunctional enzymes. They comprise a tandem arrangement of Type I-like domains, like other Type IIC enzymes (those with a fusion of a REase and MTase domains), e.g. TspGWI, TaqII and MmeI, but their sequences are only remotely similar to these previously characterized enzymes. The characterization of TspDTI, a prototype member of this group, extends our understanding of sequence-function relationships among multifunctional restriction-modification enzymes.

Background

Restriction-modification systems are a diverse class of enzymes. They are classified into four major types: I, II, III and IV. We have previously proposed the existence of a Thermus sp. enzyme family, which belongs to type II restriction endonucleases (REases), however, it features also some characteristics of types I and III. Members include related thermophilic endonucleases: TspGWI, TaqII, TspDTI, and Tth111II.

Results

Here we describe cloning, mutagenesis and analysis of the prototype TspGWI enzyme that recognises the 5'-ACGGA-3' site and cleaves 11/9 nt downstream. We cloned, expressed, and mutagenised the tspgwi gene and investigated the properties of its product, the bifunctional TspGWI restriction/modification enzyme. Since TspGWI does not cleave DNA completely, a cloning method was devised, based on amino acid sequencing of internal proteolytic fragments. The deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the Thermus sp. family – TaqII. Interestingly, these enzymes recognise similar, yet different sequences in the DNA. Both enzymes cleave DNA at the same distance, but differ in their ability to cleave single sites and in the requirement of S-adenosylmethionine as an allosteric activator for cleavage. Both the restriction endonuclease (REase) and methyltransferase (MTase) activities of wild type (wt) TspGWI (either recombinant or isolated from Thermus sp.) are dependent on the presence of divalent cations.

Conclusion

TspGWI is a bifunctional protein comprising a tandem arrangement of Type I-like domains; particularly noticeable is the central HsdM-like module comprising a helical domain and a highly conserved S-adenosylmethionine-binding/catalytic MTase domain, containing DPAVGTG and NPPY motifs. TspGWI also possesses an N-terminal PD-(D/E)XK nuclease domain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependent translocase module of the HsdR subunit and the additional domains that are involved in subunit-subunit interactions in Type I systems. The MTase and REase activities of TspGWI are autonomous and can be uncoupled. Structurally and functionally, the TspGWI protomer appears to be a streamlined 'half' of a Type I enzyme.

The TspDTI restriction endonuclease, which shows a novel recognition specificity 5′-ATGAA(N11/9)-3′, was isolated from Thermus sp. DT. TspDTI appears to be a ‘twin’ of restriction endonuclease TspGWI from Thermus sp. GW, as we have previously reported. TspGWI was isolated from the same location as TspDTI, it recognizes a related sequence 5′-ACGGA(N11/9)-3′ and has conserved cleavage positions. Both enzymes resemble two other class-IIS endonucleases from Thermus sp.: TaqII and Tth111II. N-terminal amino acid sequences of TspGWI tryptic peptides exhibit 88.9–100% similarity to the TaqII sequence. All four enzymes were purified to homogeneity; their polypeptide sizes (114.5–122 kDa) make them the largest class-IIS restriction endonucleases known to date. The existence of a Thermus sp. sub-family of class-IIS restriction endonucleases of a common origin is herein proposed.

A novel prototype class-IIS restriction endonuclease, TspGWI, was isolated from the thermophilic bacterium Thermus sp. GW. The recognition sequence and cleavage positions have been established: TspGWI recognizes the non-palindromic 5-bp sequence 5′-ACGGA-3′ and cleaves the DNA 11 and 9 nt downstream in the top and bottom strand, respectively. In addition, an accompanying endonuclease, TspGWII, an isoschizomer of Pst I, was found in Thermus sp. GW cells.

A type II restriction endonuclease activity free of TaqI was prepared from Thermus Aquaticus YT. The fraction contains two endonucleolytic components with apparently different specificities, however the major activity is sufficiently dominant to allow partial digestion analysis of the position of recognition sites. A precise determination of the location of cleavage sites in pBR322 DNA and a computer-aided search for regions of homology in the vicinity of the cut sites indicate that this enzyme recognizes the nonpalindromic sequences GACCGA or CACCCA. Other related sequences are not cleaved, in particular, GACCCA and CACCGA, indicating that the enzyme requires the identity of nucleotides in the first and fifth positions, a type of specificity that has not been previously reported. The position of cleavage is located outside of the site and is represented as: (Formula: see text).

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One hundred and forty eight isolates of the genus Thermus, from neutral and alkaline hot water springs on four continents, have been screened for the presence of restriction endonuclease activity. An isolate (SM49) from the island of Sao Miguel, in the Azores, showed a high level of restriction endonuclease activity when a cell-free extract was incubated with lambda phage DNA at 65 degrees C. A Type II restriction endonuclease (Tsp49I) has been partially purified from this isolate and the recognition and cleavage site determined. Tsp49I recognizes the four base sequence ACGT, which is the same as the recognition sequence of the mesophilic Type II restriction endonuclease MaeII. However, unlike MaeII, which cleaves DNA between the first and second bass of the recognition sequence (A/CGT), Tsp49I hydrolyses the phosphodiester bond in both strands of the substrate after the last base of the recognition sequence 5'-ACGT/-3', producing four base 3'-OH overhangs (sticky ends). The enzyme has a pH optimum of 9.0, requires 2 mM MgCl2 for maximum activity and retains full enzyme activity following incubation for 10 min at temperatures up to 8O degrees C. Two further examples of the same restriction endonuclease specificity as Tsp491 were detected in Thermus isolates from Iceland (TspIDSI) and New Zealand (TspWAM8AI). The three MaeII neoschizomers, Tsp49I, TspIDSI and TspWAM8AI, exhibit similar pH optima, heat stabilities and MgCl2 requirements, but differ in their requirements for NaCl and KCl.

127 isolates of the genus Thermus, from neutral and alkaline hot water springs on four continents, have been screened for the presence of restriction endonuclease activity. An isolate (YS52) from Yellowstone National Park, USA, showed a high level of restriction endonuclease activity when a cell free extract was incubated with lambda phage DNA at 65 degrees C. A Type II restriction endonuclease (Taq52 I) has been partially purified from this isolate and the recognition and cleavage site determined. Taq52 I has a novel interrupted palindromic tetranucleotide recognition sequence GCWGC, where W can be either adenine (A) or thymine (T). It hydrolyses the phosphodiester bond in both strands of the substrate between the first and second bases of the recognition sequence: 5'G decreased or reduced CWGC3', producing three-base 5'-OH overhangs (sticky ends). The enzyme has a pH optimum of 7.0, requires 8 mM MgCl2 for maximum activity and is thermally stable, retaining full enzyme activity following incubation at 79 degrees C for 10 min. Taq52 I not only represents a new addition to the Type II restriction endonucleases, but also increases the small list of thermostable restriction endonucleases.

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A method for detecting Type II modification methylases and determining their methylation site by assaying the ability of methylated DNA to be cleaved by heterologous restriction enzymes is described and applied to the isolation of the restriction modification methylases from Thermus thermophilus HB8, Thermus aquaticus YTI and Caryophanon latum L. M.TaqI is shown to have a methylation specificity identical to M.ThI (TCGmeA). M.ClaI methylates at adenine and protects a subset of TthI sites indicating that it methylates the sequence ATCGmeAT. Methylation by M.ThI also protects against cleavage by SalI, XhoI and at some HindII, AccI and MboI sites.

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Restriction endonucleases (13 out of 18 species used for the test) were certified to cleave single-stranded(ss)DNA. Such enzymes as AvaII, HaeII, DdeI, AluI, Sau3AI, AccII,TthHB8I and HapII were newly reported to cleave ssDNA. A model to account for the cleavage of ssDNA by restriction enzymes was proposed with supportive data. The essential part of the model was that restriction enzymes preferentially cleave transiently formed secondary structures (called canonical structures) in ssDNA composed of two recognition sequences with two fold rotational symmetry. This means that a restriction enzyme can cleave ssDNAs in general so far as the DNAs have the sequences of restriction sites for the enzyme, and that the rate of cleavage depends on the stabilities of canonical structures.

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A site specific endonuclease with novel specificity has been isolated from Thermus thermophilus strain 111 and named Tth111I. Tth111I cleaves lambda DNA into three fragments of 23.5, 25.7 and 50.8% of the total length, and ColE1 DNA into two fragments of nearly equal length. The sequences around Tth111I cleavage sites of ColE1 and lambda DNA were determined by the Maxam and Gilbert method and the two dimensional mapping method. The results suggest that Tth111I recognizes the DNA sequence (formula: see text) and cleaves the site as indicated by the arrows. Assuming that the first T.A pair in the sequence can be replaced for any base pair, the Tth111I recognition sequence has the symmetry with the two-fold axis as most type II restriction endonucleases do.

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A second site specific endonuclease with novel specificity has been purified from Thermus thermophilus strain 111 and named Tth111II. The enzyme is active at temperature up to 80 degrees C and requires Mg2+ or Mn2+ for endonuclease activity. Tth111II cleaves phi X174RFDNA into 11 fragments and lambda NA into more than 25 fragments. From the 5'-terminal sequences of TthlllII fragments of phi X174RFDNA determined by the two dimensional homochromatography and the survey on nucleotide sequence of phi X174RFDNA, it was concluded that Tth111II recognizes the DNA sequence (see former index) and cleaves the sites as indicated by the arrows.

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In an extensive analysis, using a range of restriction endonucleases, HinfI and TaqI were found to differentiate satellites I, II and III & IV. Satellite I is resistant to digestion by TaqI, but is cleaved by HinfI to yield three major fragments of approximate size 770, 850 and 950bp, associated in a single length of DNA. The 770bp fragment contains recognition sites for a number of other enzymes, whereas the 850 and 950bp fragments are "silent" by restriction enzyme analysis. Satellite II is digested by HinfI into a large number of very small (10-80bp) fragments, many of which also contain TaqI sites. A proportion of the HinfI sites in satellite II have the sequence 5'GA(GC)TC. The HinfI digestion products of satellites III and IV form a complete ladder, stretching from 15bp or less to more than 250bp, with adjacent multimers separated by an increment of 5bp. The ladder fragments do not contain TaqI sites and all HinfI sites have the sequence 5'GA(AT)TC. Three fragments from the HinfI ladder of satellite III have been sequenced, and all consist of a tandemly repeated 5bp sequence, 5'TTCCA, with a non-repeated, G+C rich sequence, 9bp in length, at the 3' end.

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The Taq I modification and restriction genes (recognition sequence TCGA) have been cloned in E. coli and their DNA sequences have been determined. Both proteins were characterized and the N-terminal sequence of the endonuclease was determined. The genes have the same transcriptional orientation with the methylase gene 5' to the endonuclease gene. The methylase gene is 1089 bp in length (363 amino acids, 40,576 daltons); the endonuclease gene is 702 bp in length (234 amino acids, 27,523 daltons); they are separated by 132 bp. Both methylase and endonuclease activity can be detected in cell extracts. The clones fully modify the vector and chromosomal DNA but they fail to restrict infecting phage. Clones carrying only the restriction gene are viable even in the absence of modification. The restriction gene contains 7 Taq I sites; the modification gene contains none. This asymmetric distribution of sites could be important in the regulation of the expression of the endonuclease gene.

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Orthodox Type IIP restriction endonucleases, which are commonly used in molecular biological work, recognize a single palindromic DNA recognition sequence and cleave within or near this sequence. Several new studies have reported on structural and biochemical peculiarities of restriction endonucleases that differ from the orthodox in that they require two copies of a particular DNA recognition sequence to cleave the DNA. These two sites requiring restriction endonucleases belong to different subtypes of Type II restriction endonucleases, namely Types IIE, IIF and IIS. We compare enzymes of these three types with regard to their DNA recognition and cleavage properties. The simultaneous recognition of two identical DNA sites by these restriction endonucleases ensures that single unmethylated recognition sites do not lead to chromosomal DNA cleavage, and might reflect evolutionary connections to other DNA processing proteins that specifically function with two sites.

Restriction endonucleases HindII and TaqI, but not SalI, were found to efficiently cleave synthetic hexadecanucleotide duplexes which contained either an A/C or a G/T mismatch within their respective restriction sites. Double-stranded M13 DNAs with identical mismatches were also cleaved under the assay conditions. These results suggest that the distortion of the DNA duplex, caused by these purine/pyrimidine mismatches is not sufficiently large so as to interfere with the recognition and the subsequent cleavage of the DNA by these two enzymes. HindII and SalI, but not TaqI, were furthermore shown to hydrolyze the two strands of the duplex with different rates. The differences between the mode of recognition of their respective restriction sites by these three enzymes are discussed.

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The modified base alpha-putrescinylthymine (putT) in phi W-14 DNA blocks cleavage of the DNA by 17 of 32 Type II restriction endonucleases. The enzymes cleaving the DNA do so to widely varying extents. The frequencies of cleavage of three altered forms of the DNA show that putT blocks recognition sites either when it occurs within the site or when it is in a sequence flanking the site. The blocking is dependent on both charge and steric factors. The charge effects can be greater than the steric effects for some of the enzymes tested. All the enzymes cleaving phi W-14 DNA release discrete fragments, showing that the distribution of putT is ordered. The cleavage frequencies for different enzymes suggest that the sequence CAputTG occurs frequently in the DNA. Only TaqI of the enzymes tested appeared not to be blocked by putT, but it was slowed down. TaqI generated fragments are joinable by T4 DNA ligase.

Synthetic hexadecanucleotide duplexes containing a single purine nucleotide analogue in the recognition sites of the restriction endonucleases HindII, SalI and TaqI were used to investigate the restriction site determinants required by these enzymes for sequence recognition and phosphodiester bond cleavage. The enzymes were, in general, unaffected by changes introduced into the minor groove of the helix. SalI was found to be inhibited by the major groove modifications introduced into the fourth position of its recognition sequence GTCGAC. HindII and TaqI were, by contrast, able to cleave the sites containing the analogues at this position. TaqI and, to a lesser extent, HindII could also be shown to tolerate "mismatch analogues" at this site.

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.

Adenovirus type 3 (Ad3) isolates, isolated from 45 patients with acute conjunctivitis during the year 1990 in Japan, were studied by DNA restriction enzyme analysis with restriction endonucleases recognizing 6-bp sequences (BamHI, SmaI, HindIII, BglII) and endonucleases recognizing 5- or 4-bp sequences (HinfI and TaqI). All 45 isolates of Ad3 were identified as the genome type Ad3f by six endonucleases. They were further classified into three varieties by HinfI, varieties H1 (87.5%), H2 (8.9%), and H3 (2.2%), and into five varieties by TaqI, varieties T1 (75.6%), T2 (13.3%), T3 (2.2%), T4 (4.5%), and T5 (8.9%). The use of HinfI and TaqI was sufficient to distinguish six subgenome types: types Ad3fH1T1, Ad3fH1T2, Ad3fH1T4, Ad3fH1T5, Ad3fH2T1, and Ad3fH3T3. Among them, Ad3fH1T1 was predominant in areas where the virus is epidemic. The study showed that the same Ad3 genome type, genome type Ad3f, caused acute viral conjunctivitis even in different areas in Japan. The source and the route of infection were suggested to be common in areas where the virus is epidemic.

A type IIG restriction endonuclease, RM.BpuSI from Bacillus pumilus, has been characterized and its X-ray crystal structure determined at 2.35Å resolution. The enzyme is comprised of an array of 5-folded domains that couple the enzyme's N-terminal endonuclease domain to its C-terminal target recognition and methylation activities. The REase domain contains a PD-x15-ExK motif, is closely superimposable against the FokI endonuclease domain, and coordinates a single metal ion. A helical bundle domain connects the endonuclease and methyltransferase (MTase) domains. The MTase domain is similar to the N6-adenine MTase M.TaqI, while the target recognition domain (TRD or specificity domain) resembles a truncated S subunit of Type I R–M system. A final structural domain, that may form additional DNA contacts, interrupts the TRD. DNA binding and cleavage must involve large movements of the endonuclease and TRD domains, that are probably tightly coordinated and coupled to target site methylation status.

Type I restriction endonucleases are intriguing, multifunctional complexes that restrict DNA randomly, at sites distant from the target sequence. Restriction at distant sites is facilitated by ATP hydrolysis-dependent, translocation of double-stranded DNA towards the stationary enzyme bound at the recognition sequence. Following restriction, the enzymes are thought to remain associated with the DNA at the target site, hydrolyzing copious amounts of ATP. As a result, for the past 35 years type I restriction endonucleases could only be loosely classified as enzymes since they functioned stoichiometrically relative to DNA. To further understand enzyme mechanism, a detailed analysis of DNA cleavage by the EcoR124I holoenzyme was done. We demonstrate for the first time that type I restriction endonucleases are not stoichiometric but are instead catalytic with respect to DNA. Further, the mechanism involves formation of a dimer of holoenzymes, with each monomer bound to a target sequence and, following cleavage, each dissociates in an intact form to bind and restrict subsequent DNA molecules. Therefore, type I restriction endonucleases, like their type II counterparts, are true enzymes. The conclusion that type I restriction enzymes are catalytic relative to DNA has important implications for the in vivo function of these previously enigmatic enzymes.

From Caryophanon latum L site specific restriction endonuclease (ClaI) has been purified, which recognises tha DNA hexanucleotide palindrome 5'-A-T-C-G-A-T-3'. Staggered cleavage generates DNA restriction fragments with 5'-terminal pCG extensions. A CLaI map of bacteriophage lambda has been determined, which indicates cleavage inhibition due to adenine methylation at over lapping ClaI-GATC recognition sequences. Plasmid pBR322 is cut only once, in the tetracycline promoter region, and can, therefore, be used as a vector system for cloning fragments derived from ClaI digestions, and in addition for fragments generated by TaqI, HpaII, and several other enzymes.

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MmeI is an unusual Type II restriction enzyme that is useful for generating long sequence tags. We have cloned the MmeI restriction-modification (R-M) system and found it to consist of a single protein having both endonuclease and DNA methyltransferase activities. The protein comprises an amino-terminal endonuclease domain, a central DNA methyltransferase domain and C-terminal DNA recognition domain. The endonuclease cuts the two DNA strands at one site simultaneously, with enzyme bound at two sites interacting to accomplish scission. Cleavage occurs more rapidly than methyl transfer on unmodified DNA. MmeI modifies only the adenine in the top strand, 5′-TCCRAC-3′. MmeI endonuclease activity is blocked by this top strand adenine methylation and is unaffected by methylation of the adenine in the complementary strand, 5′-GTYGGA-3′. There is no additional DNA modification associated with the MmeI R-M system, as is required for previously characterized Type IIG R-M systems. The MmeI R-M system thus uses modification on only one of the two DNA strands for host protection. The MmeI architecture represents a minimal approach to assembling a restriction-modification system wherein a single DNA recognition domain targets both the endonuclease and DNA methyltransferase activities.

We describe the characterisation of four thermostable NAD+-dependent DNA ligases, from Thermus thermophilus (Tth), Thermus scotoductus (Ts), Rhodothermus marinus (Rm) and Thermus aquaticus (Taq), by an assay which measures ligation rate and mismatch discrimination. Complete libraries of octa-, nona- and decanucleotides were used as substrates. The assay comprised the polymerisation of oligonucleotides initiated from a 17 base ‘primer’, using M13mp18 ssDNA as template. Polymers of ligation products were analysed by polyacrylamide gel electrophoresis. Under optimum conditions, the enzymes produced polymers ranging from 8 to 16 additions; there was variation between enzymes and the length of the oligonucleotides had a strong effect. The optimal total oligonucleotide concentration for each library was ~4 nmol. We compared the rates of ligation between the four ligases using an octanucleotide library as substrate. By this criterion, the Ts and Rm ligases are far more active compared to the more commonly available thermostable ligases.

Restriction endonucleases are indispensable tools in molecular biology and biotechnology. Type II restriction endonucleases are part of restriction modification systems. DNA fragment extraction and restriction mapping are the basis for several biotechnological activities. WebFARM is a server application for identifying restriction endonuclease recognition sites and to give information regarding restriction mapping for given nucleotide sequences. WebFARM analyses given nucleotide sequence and identify restriction site for selected restriction endonucleases. It will also provide frequency of restriction for each restriction endonuclease.

Availability

http://webfarm.bioinfoindia.org/