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1.  Three-stage biochemical selection: cloning of prototype class IIS/IIC/IIG restriction endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus 
BMC Molecular Biology  2013;14:17.
In continuing our research into the new family of bifunctional restriction endonucleases (REases), we describe the cloning of the tsoIRM gene. Currently, the family includes six thermostable enzymes: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI, TsoI, isolated from various Thermus sp. and two thermolabile enzymes: RpaI and CchII, isolated from mesophilic bacteria Rhodopseudomonas palustris and Chlorobium chlorochromatii, respectively. The enzymes have several properties in common. They are large proteins (molecular size app. 120 kDa), coded by fused genes, with the REase and methyltransferase (MTase) in a single polypeptide, where both activities are affected by S-adenosylmethionine (SAM). They recognize similar asymmetric cognate sites and cleave at a distance of 11/9 nt from the recognition site. Thus far, we have cloned and characterised TaqII, Tth111II, TthHB27I, TspGWI and TspDTI.
TsoI REase, which originate from thermophilic Thermus scotoductus RFL4 (T. scotoductus), was cloned in Escherichia coli (E. coli) using two rounds of biochemical selection of the T. scotoductus genomic library for the TsoI methylation phenotype. DNA sequencing of restriction-resistant clones revealed the common open reading frame (ORF) of 3348 bp, coding for a large polypeptide of 1116 aminoacid (aa) residues, which exhibited a high level of similarity to Tth111II (50% identity, 60% similarity). The ORF was PCR-amplified, subcloned into a pET21 derivative under the control of a T7 promoter and was subjected to the third round of biochemical selection in order to isolate error-free clones. Induction experiments resulted in synthesis of an app. 125 kDa protein, exhibiting TsoI-specific DNA cleavage. Also, the wild-type (wt) protein was purified and reaction optima were determined.
Previously we identified and cloned the Thermus family RM genes using a specially developed method based on partial proteolysis of thermostable REases. In the case of TsoI the classic biochemical selection method was successful, probably because of the substantially lower optimal reaction temperature of TsoI (app. 10-15°C). That allowed for sufficient MTase activity in vivo in recombinant E. coli. Interestingly, TsoI originates from bacteria with a high optimum growth temperature of 67°C, which indicates that not all bacterial enzymes match an organism’s thermophilic nature, and yet remain functional cell components. Besides basic research advances, the cloning and characterisation of the new prototype REase from the Thermus sp. family enzymes is also of practical importance in gene manipulation technology, as it extends the range of available DNA cleavage specificities.
PMCID: PMC3751577  PMID: 23919831
2.  Modified ‘one amino acid-one codon’ engineering of high GC content TaqII-coding gene from thermophilic Thermus aquaticus results in radical expression increase 
An industrial approach to protein production demands maximization of cloned gene expression, balanced with the recombinant host’s viability. Expression of toxic genes from thermophiles poses particular difficulties due to high GC content, mRNA secondary structures, rare codon usage and impairing the host’s coding plasmid replication.
TaqII belongs to a family of bifunctional enzymes, which are a fusion of the restriction endonuclease (REase) and methyltransferase (MTase) activities in a single polypeptide. The family contains thermostable REases with distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI and a few enzymes found in mesophiles. While not being isoschizomers, the enzymes exhibit amino acid (aa) sequence homologies, having molecular sizes of ~120 kDa share common modular architecture, resemble Type-I enzymes, cleave DNA 11/9 nt from the recognition sites, their activity is affected by S-adenosylmethionine (SAM).
We describe the taqIIRM gene design, cloning and expression of the prototype TaqII. The enzyme amount in natural hosts is extremely low. To improve expression of the taqIIRM gene in Escherichia coli (E. coli), we designed and cloned a fully synthetic, low GC content, low mRNA secondary structure taqIIRM, codon-optimized gene under a bacteriophage lambda (λ) P R promoter. Codon usage based on a modified ‘one amino acid–one codon’ strategy, weighted towards low GC content codons, resulted in approximately 10-fold higher expression of the synthetic gene. 718 codons of total 1105 were changed, comprising 65% of the taqIIRM gene. The reason for we choose a less effective strategy rather than a resulting in high expression yields ‘codon randomization’ strategy, was intentional, sub-optimal TaqII in vivo production, in order to decrease the high ‘toxicity’ of the REase-MTase protein.
Recombinant wt and synthetic taqIIRM gene were cloned and expressed in E. coli. The modified ‘one amino acid–one codon’ method tuned for thermophile-coded genes was applied to obtain overexpression of the ‘toxic’ taqIIRM gene. The method appears suited for industrial production of thermostable ‘toxic’ enzymes in E. coli. This novel variant of the method biased toward increasing a gene’s AT content may provide economic benefits for industrial applications.
PMCID: PMC3893498  PMID: 24410856
3.  Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving 
BMC Biochemistry  2011;12:62.
The TaqII enzyme is a member of the Thermus sp. enzyme family that we propounded previously within Type IIS restriction endonucleases, containing related thermophilic bifunctional endonucleases-methyltransferases from various Thermus sp.: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI and TsoI. These enzymes show significant nucleotide and amino acid sequence similarities, a rare phenomenon among restriction endonucleases, along with similarities in biochemical properties, molecular size, DNA recognition sequences and cleavage sites. They also feature some characteristics of Types I and III.
Barker et al. reported the Type IIS/IIC restriction endonuclease TaqII as recognizing two distinct cognate site variants (5'-GACCGA-3' and 5'-CACCCA-3') while cleaving 11/9 nucleotides downstream. We used four independent methods, namely, shotgun cloning and sequencing, restriction pattern analysis, digestion of particular custom substrates and GeneScan analysis, to demonstrate that the recombinant enzyme recognizes only 5'-GACCGA-3' sites and cleaves 11/9 nucleotides downstream. We did not observe any 5'-CACCCA-3' cleavage under a variety of conditions and site arrangements tested. We also characterized the enzyme biochemically and established new digestion conditions optimal for practical enzyme applications. Finally, we developed and propose a new version of the Fidelity Index - the Fidelity Index for Partial Cleavage (FI-PC).
The DNA recognition sequence of the bifunctional prototype TaqII endonuclease-methyltransferase from Thermus aquaticus has been redefined as recognizing only 5'-GACCGA-3' cognate sites. The reaction conditions (pH and salt concentrations) were designed either to minimize (pH = 8.0 and 10 mM ammonium sulphate) or to enhance star activity (pH = 6.0 and no salt). Redefinition of the recognition site and reaction conditions makes this prototype endonuclease a useful tool for DNA manipulation; as yet, this enzyme has no practical applications. The extension of the Fidelity Index will be helpful for DNA manipulation with enzymes only partially cleaving DNA.
PMCID: PMC3280180  PMID: 22141927
4.  A new Thermus sp. class-IIS enzyme sub-family: isolation of a ‘twin’ endonuclease TspDTI with a novel specificity 5′-ATGAA(N11/9)-3′, related to TspGWI, TaqII and Tth111II 
Nucleic Acids Research  2003;31(14):e74.
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.
PMCID: PMC167652  PMID: 12853651
5.  Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family 
BMC Molecular Biology  2009;10:52.
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.
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.
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.
PMCID: PMC2700111  PMID: 19480701
6.  Cofactor analogue-induced chemical reactivation of endonuclease activity in a DNA cleavage/methylation deficient TspGWI N473A variant in the NPPY motif 
Molecular Biology Reports  2014;41:2313-2323.
We reported previously that TspGWI, a prototype enzyme of a new Thermus sp. family of restriction endonucleases-methyltransferases (REases-MTases), undergoes the novel phenomenon of sinefungin (SIN)-caused specificity transition. Here we investigated mutant TspGWI N473A, containing a single amino acid (aa) substitution in the NPPY motif of the MTase. Even though the aa substitution is located within the MTase polypeptide segment, DNA cleavage and modification are almost completely abolished, indicating that the REase and MTase are intertwined. Remarkably, the TspGWI N473A REase functionality can be completely reconstituted by the addition of SIN. We hypothesize that SIN binds specifically to the enzyme and restores the DNA cleavage-competent protein tertiary structure. This indicates the significant role of allosteric effectors in DNA cleavage in Thermus sp. enzymes. This is the first case of REase mutation suppression by an S-adenosylmethionine (SAM) cofactor analogue. Moreover, the TspGWI N473A clone strongly affects E. coli division control, acting as a ‘selfish gene’. The mutant lacks the competing MTase activity and therefore might be useful for applications in DNA manipulation. Here we present a case study of a novel strategy for REase activity/specificity alteration by a single aa substitution, based on the bioinformatic analysis of active motif locations, combining (a) aa sequence engineering (b) the alteration of protein enzymatic properties, and (c) the use of cofactor–analogue cleavage reconstitution and stimulation.
PMCID: PMC3968444  PMID: 24442320
Endonuclease-methyltransferase; Thermus sp. enzyme; Enzymatic reaction cofactor; Cofactor analogue; Sinefungin; S-adenosylmethione; Mutant activation; Specificity change
7.  Characterization of cleavage intermediate and star sites of RM.Tth111II 
Scientific Reports  2014;4:3838.
Tth111II is a thermostable Type IIGS restriction enzyme that recognizes DNA sites CAARCA (R = A or G) and cleaves downstream at N11/N9. Here, the tth111IIRM gene was cloned and expressed in E. coli, and Tth111II was purified. The purified enzyme contains internally-bound S-adenosylmethionine (SAM). When the internal SAM was removed, the endonuclease activity was stimulated by adding SAM or its analog sinefungin. The cleavage intermediate is mostly top-strand nicked DNA on a single-site plasmid. Addition of duplex oligos with a cognate site stimulates cleavage activity of the one-site substrate. Tth111II cleaves a two-site plasmid DNA with equal efficiency regardless of site orientation. We propose the top-strand nicking is carried out by a Tth111II monomer and bottom-strand cleavage is carried out by a transient dimer. Tth111II methylates cleavage product-like duplex oligos CAAACAN9, but the modification rate is estimated to be much slower than the top-strand nicking rate. We cloned and sequenced a number of Tth111II star sites which are 1-bp different from the cognate sites. A biochemical pathway is proposed for the restriction and methylation activities of Tth111II.
PMCID: PMC3899748  PMID: 24452415
8.  TspGWI, a thermophilic class-IIS restriction endonuclease from Thermus sp., recognizes novel asymmetric sequence 5′-ACGGA(N11/9)-3′ 
Nucleic Acids Research  2002;30(7):e33.
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.
PMCID: PMC101857  PMID: 11917039
9.  A spite specific endonuclease from thermus thermophilus 111, Tth111I. 
Nucleic Acids Research  1980;8(1):43-56.
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.
PMCID: PMC327241  PMID: 6243779
10.  A new genomic tool, ultra-frequently cleaving TaqII/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse DNA libraries 
BMC Genomics  2013;14:370.
Genomics and metagenomics are currently leading research areas, with DNA sequences accumulating at an exponential rate. Although enormous advances in DNA sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. A number of DNA fragmentation methods, such as hydrodynamic sharing, sonication or DNase I fragmentation, have various drawbacks, including DNA damage, poor fragmentation control, irreproducibility and non-overlapping DNA segment representation. Improvements in these limited DNA scission methods are consequently needed. An alternative method for obtaining higher quality DNA fragments involves partial digestion with restriction endonucleases (REases).
We have shown previously that class-IIS/IIC/IIG TspGWI REase, the prototype member of the Thermus sp. enzyme family, can be chemically relaxed by a cofactor analogue, allowing it to recognize very short DNA sequences of 3-bp combined frequency. Such frequently cleaving REases are extremely rare, with CviJI/CviJI*, SetI and FaiI the only other ones found in nature. Their unusual features make them very useful molecular tools for the development of representative DNA libraries.
We constructed a horse genomic library and a deletion derivative library of the butyrylcholinesterase cDNA coding region using a novel method, based on TaqII, Thermus sp. family bifunctional enzyme exhibiting cofactor analogue specificity relaxation. We used sinefungin (SIN) – an S-adenosylmethionine (SAM) analogue with reversed charge pattern, and dimethylsulfoxide (DMSO), to convert the 6-bp recognition site TaqII (5′-GACCGA-3′ [11/9]) into a theoretical 2.9-bp REase, with 70 shortened variants of the canonical recognition sequence detected. Because partial DNA cleavage is an inherent feature of the Thermus sp. enzyme family, this modified TaqII is uniquely suited to quasi-random library generation.
In the presence of SIN/DMSO, TaqII REase is transformed from cleaving every 4096 bp on average to cleaving every 58 bp. TaqII SIN/DMSO thus extends the palette of available REase prototype specificities. This phenomenon, employed under partial digestion conditions, was applied to quasi-random DNA fragmentation. Further applications include high sensitivity probe generation and metagenomic DNA amplification.
PMCID: PMC3681635  PMID: 23724933
11.  Type II restriction endonucleases cleave single-stranded DNAs in general. 
Nucleic Acids Research  1985;13(16):5747-5760.
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.
PMCID: PMC321909  PMID: 2994012
12.  Real-time kinetics of restriction–modification gene expression after entry into a new host cell 
Nucleic Acids Research  2008;36(8):2581-2593.
Most type II restriction–modification (R–M) systems produce separate restriction endonuclease (REase) and methyltransferase (MTase) proteins. After R–M system genes enter a new cell, protective MTase must appear before REase to avoid host chromosome cleavage. The basis for this apparent temporal regulation is not well understood. PvuII and some other R–M systems appear to achieve this delay by cotranscribing the REase gene with the gene for an autogenous transcription activator/repressor (the ‘C’ protein C.PvuII). To test this model, bacteriophage M13 was used to introduce the PvuII genes into a bacterial population in a relatively synchronous manner. REase mRNA and activity appeared ∼10 min after those of the MTase, but never rose if there was an inactivating pvuIIC mutation. Infection with recombinant M13pvuII phage had little effect on cell growth, relative to infection with parental M13. However, infection of cells pre-expressing C.PvuII led to cessation of growth. This study presents the first direct demonstration of delayed REase expression, relative to MTase, when type II R–M genes enter a new host cell. Surprisingly, though the C and REase genes are cotranscribed, the pvuIIC portion of the mRNA was more abundant than the pvuIIR portion after stable establishment of the R–M system.
PMCID: PMC2377437  PMID: 18334533
13.  A second site specific endonuclease from Thermus thermophilus 111, Tth111II. 
Nucleic Acids Research  1980;8(15):3275-3285.
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.
PMCID: PMC324152  PMID: 6255411
14.  The Role of the Methyltransferase Domain of Bifunctional Restriction Enzyme RM.BpuSI in Cleavage Activity 
PLoS ONE  2013;8(11):e80967.
Restriction enzyme (REase) RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS), bifunctional polypeptide possessing both methyltransferase (MTase) and endonuclease activities (Type IIC) and endonuclease activity stimulated by S-adenosyl-L-methionine (SAM) (Type IIG). The stimulatory effect of SAM on cleavage activity presents a major paradox: a co-factor of the MTase activity that renders the substrate unsusceptible to cleavage enhances the cleavage activity. Here we show that the RM.BpuSI MTase activity modifies both cleavage substrate and product only when they are unmethylated. The MTase activity is, however, much lower than that of M1.BpuSI and is thought not to be the major MTase for host DNA protection. SAM and sinefungin (SIN) increase the Vmax of the RM.BpuSI cleavage activity with a proportional change in Km, suggesting the presence of an energetically more favorable pathway is taken. We further showed that RM.BpuSI undergoes substantial conformational changes in the presence of Ca2+, SIN, cleavage substrate and/or product. Distinct conformers are inferred as the pre-cleavage/cleavage state (in the presence of Ca2+, substrate or both) and MTase state (in the presence of SIN and substrate, SIN and product or product alone). Interestingly, RM.BpuSI adopts a unique conformation when only SIN is present. This SIN-bound state is inferred as a branch point for cleavage and MTase activity and an intermediate to an energetically favorable pathway for cleavage, probably through increasing the binding affinity of the substrate to the enzyme under cleavage conditions. Mutation of a SAM-binding residue resulted in altered conformational changes in the presence of substrate or Ca2+ and eliminated cleavage activity. The present study underscores the role of the MTase domain as facilitator of efficient cleavage activity for RM.BpuSI.
PMCID: PMC3817140  PMID: 24224063
15.  Structural asymmetry in the Thermus thermophilus RuvC dimer suggests a basis for sequential strand cleavages during Holliday junction resolution 
Nucleic Acids Research  2012;41(1):648-656.
Holliday junction (HJ) resolvases are structure-specific endonucleases that cleave four-way DNA junctions (HJs) generated during DNA recombination and repair. Bacterial RuvC, a prototypical HJ resolvase, functions as homodimer and nicks DNA strands precisely across the junction point. To gain insights into the mechanisms underlying symmetrical strand cleavages by RuvC, we performed crystallographic and biochemical analyses of RuvC from Thermus thermophilus ( RuvC). The crystal structure of RuvC shows an overall protein fold similar to that of Escherichia coli RuvC, but RuvC has a more tightly associated dimer interface possibly reflecting its thermostability. The binding mode of a HJ-DNA substrate can be inferred from the shape/charge complementarity between the RuvC dimer and HJ-DNA, as well as positions of sulfate ions bound on the protein surface. Unexpectedly, the structure of RuvC homodimer refined at 1.28 Å resolution shows distinct asymmetry near the dimer interface, in the region harboring catalytically important aromatic residues. The observation suggests that the RuvC homodimer interconverts between two asymmetric conformations, with alternating subunits switched on for DNA strand cleavage. This model provides a structural basis for the ‘nick-counter-nick’ mechanism in HJ resolution, a mode of HJ processing shared by prokaryotic and eukaryotic HJ resolvases.
PMCID: PMC3592405  PMID: 23118486
16.  A new restriction endonuclease from Spirulina platensis. 
Nucleic Acids Research  1986;14(5):1985-1989.
Three restriction endonucleases, Sp1I, Sp1II and Sp1III have been purified partially from Spirulina platensis subspecies siamese and named. Sp1I cleaves bacteriophage lambda DNA at one site, phi X 174 RF DNA at two sites, but does not cleave pBR322 DNA. This enzyme recognizes the sequence 5'CGTACG3' 3'GCATCG5' and cuts the site indicated by the arrows. Sp1II is an isoschizomer of Tth111I and Sp1III is an isoschizomer of HaeIII.
PMCID: PMC339637  PMID: 3008081
17.  Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system 
Nucleic Acids Research  2009;37(3):983-998.
Most type II restriction-modification (R-M) systems produce separate endonuclease (REase) and methyltransferase (MTase) proteins. After R-M genes enter a new cell, MTase activity must appear before REase or the host chromosome will be cleaved. Temporal control of these genes thus has life-or-death consequences. PvuII and some other R-M systems delay endonuclease expression by cotranscribing the REase gene with the upstream gene for an autogenous activator/repressor (C protein). C.PvuII was previously shown to have low levels early, but positive feedback later boosts transcription of the C and REase genes. The MTase is expressed without delay, and protects the host DNA. C.PvuII binds to two sites upstream of its gene: OL, associated with activation, and OR, associated with repression. Even when symmetry elements of each operator are made identical, C.PvuII binds preferentially to OL. In this study, the intra-operator spacers are shown to modulate relative C.PvuII affinity. In light of a recently reported C.Esp1396I-DNA co-crystal structure, in vitro and in vivo effects of altering OL and OR spacers were determined. The results suggest that the GACTnnnAGTC consensus is the primary determinant of C.PvuII binding affinity, with intra-operator spacers playing a fine-tuning role that affects mobility of this R-M system.
PMCID: PMC2647307  PMID: 19126580
18.  KpnI restriction endonuclease and methyltransferase exhibit contrasting mode of sequence recognition 
Nucleic Acids Research  2004;32(10):3148-3155.
The molecular basis of the interaction of KpnI restriction endonuclease (REase) and the corresponding methyltransferase (MTase) at their cognate recognition sequence is investigated using a range of footprinting techniques. DNase I protection analysis with the REase reveals the protection of a 14–18 bp region encompassing the hexanucleotide recognition sequence. The MTase, in contrast, protects a larger region. KpnI REase contacts two adjacent guanine residues and the single adenine residue in both the strands within the recognition sequence 5′-GGTACC-3′, inferred by dimethylsulfate (DMS) protection, interference and missing nucleotide interference analysis. In contrast, KpnI MTase does not show elaborate base-specific contacts. Ethylation interference analysis also showed the differential interaction of REase and MTase with phosphate groups of three adjacent bases on both strands within the recognition sequence. The single thymine residue within the sequence is hyper- reactive to the permanganate oxidation, consistent with MTase-induced base flipping. The REase on the other hand does not show any major DNA distortion. The results demonstrate that the differences in the molecular interaction pattern of the two proteins at the same recognition sequence reflect the contrasting chemistry of DNA cleavage and methylation catalyzed by these two dissimilar enzymes, working in combination as constituents of a cellular defense strategy.
PMCID: PMC434444  PMID: 15192117
19.  A bistable hysteretic switch in an activator–repressor regulated restriction–modification system 
Nucleic Acids Research  2013;41(12):6045-6057.
Restriction–modification (RM) systems are extremely widespread among bacteria and archaea, and are often specified by mobile genetic elements. In type II RM systems, where the restriction endonuclease (REase) and protective DNA methyltransferase (MTase) are separate proteins, a major regulatory challenge is delaying expression of the REase relative to the MTase after RM genes enter a new host cell. Basic understanding of this regulation is available for few RM systems, and detailed understanding for none. The PvuII RM system is one of a large subset in which the central regulatory role is played by an activator–repressor protein (called C, for controller). REase expression depends upon activation by C, whereas expression of the MTase does not. Thus delay of REase expression depends on the rate of C-protein accumulation. This is a nonlinear process, as C also activates transcription of its own gene. Mathematical modeling of the PvuII system led to the unexpected predictions of responsiveness to a factor not previously studied in RM system control—gene copy number—and of a hysteretic response. In this study, those predictions have been confirmed experimentally. The results may apply to many other C-regulated RM systems, and help explain their ability to spread so widely.
PMCID: PMC3695507  PMID: 23630319
20.  Naturally-occurring, dually-functional fusions between restriction endonucleases and regulatory proteins 
Restriction-modification (RM) systems appear to play key roles in modulating gene flow among bacteria and archaea. Because the restriction endonuclease (REase) is potentially lethal to unmethylated new host cells, regulation to ensure pre-expression of the protective DNA methyltransferase (MTase) is essential to the spread of RM genes. This is particularly true for Type IIP RM systems, in which the REase and MTase are separate, independently-active proteins. A substantial subset of Type IIP RM systems are controlled by an activator-repressor called C protein. In these systems, C controls the promoter for its own gene, and for the downstream REase gene that lacks its own promoter. Thus MTase is expressed immediately after the RM genes enter a new cell, while expression of REase is delayed until sufficient C protein accumulates. To study the variation in and evolution of this regulatory mechanism, we searched for RM systems closely related to the well-studied C protein-dependent PvuII RM system. Unexpectedly, among those found were several in which the C protein and REase genes were fused.
The gene for CR.NsoJS138I fusion protein (nsoJS138ICR, from the bacterium Niabella soli) was cloned, and the fusion protein produced and partially purified. Western blots provided no evidence that, under the conditions tested, anything other than full-length fusion protein is produced. This protein had REase activity in vitro and, as expected from the sequence similarity, its specificity was indistinguishable from that for PvuII REase, though the optimal reaction conditions were different. Furthermore, the fusion was active as a C protein, as revealed by in vivo activation of a lacZ reporter fusion to the promoter region for the nsoJS138ICR gene.
Fusions between C proteins and REases have not previously been characterized, though other fusions have (such as between REases and MTases). These results reinforce the evidence for impressive modularity among RM system proteins, and raise important questions about the implications of the C-REase fusions on expression kinetics of these RM systems.
PMCID: PMC3850674  PMID: 24083337
Restriction-modification systems; Restriction endonuclease; Gene regulation; Fused genes; C protein; Regulatory evolution
21.  Virion-Associated Restriction Endonucleases of Chloroviruses 
Journal of Virology  2006;80(16):8114-8123.
Chloroviruses are large, double-stranded-DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae. The prototype of the genus is Paramecium bursaria chlorella virus 1 (PBCV-1). Chlorovirus genomes contain various amounts of methylated nucleotides due to virus-encoded DNA methyltransferases (MTases); about 25% of the MTases are associated with companion DNA site-specific (restriction) endonucleases (REases). These enzymes constitute virally encoded restriction-modification (R/M) systems. Although several of the chlorovirus R/M systems are characterized, their biological functions are unknown. The PBCV-1 proteome reveals that two virus-encoded REases, but not their companion MTases, are virion associated, suggesting that viral REases might help degrade the host DNA early in infection. To test this hypothesis, host chromosomal DNA from PBCV-1-infected cells was examined by pulsed-field gel electrophoresis. Initiation of host chromosomal DNA degradation occurred within 5 min postinfection (p.i.). The DNA degradation was insensitive to protein synthesis inhibitors or UV inactivation of virus particles, consistent with the agent being a small protein associated with the virion. Nuclease activities, including those of the two predicted REases and an uncharacterized general nuclease(s), were detected in disrupted PBCV-1 particles. The general nuclease(s) degraded both host and viral DNAs in vitro, although the viral DNA was not degraded in vivo, suggesting differential intracellular trafficking of the virion-associated nucleases. Infection with chloroviruses lacking an R/M system(s) resulted in either delayed host chromosomal DNA degradation or no detectable host chromatin changes. These immediate-early events associated with chlorovirus infections may facilitate rapid switching of the host transcriptional apparatus to viral transcription, which begins within 5 to 10 min p.i.
PMCID: PMC1563800  PMID: 16873267
22.  Tsp49I (ACGT/), a thermostable neoschizomer of the Type II restriction endonuclease MaeII (A/CGT), discovered in isolates of the genus Thermus from the Azores, Iceland and New Zealand. 
Nucleic Acids Research  1996;24(10):1799-1801.
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.
PMCID: PMC145888  PMID: 8657557
23.  Regulatory circuit based on autogenous activation-repression: roles of C-boxes and spacer sequences in control of the PvuII restriction-modification system 
Nucleic Acids Research  2007;35(20):6935-6952.
Type II restriction-modification (R-M) systems comprise a restriction endonuclease (REase) and a protective methyltransferase (MTase). After R-M genes enter a new cell, MTase must appear before REase or the chromosome will be cleaved. PvuII and some other R-M systems achieve this delay by cotranscribing the REase gene with the gene for an autogenous transcription activator (the controlling or ‘C’ protein C.PvuII). This study reveals, through in vivo titration, that C.PvuII is not only an activator but also a repressor for its own gene. In other systems, this type of circuit can result in oscillatory behavior. Despite the use of identical, symmetrical C protein-binding sequences (C-boxes) in the left and right operators, C.PvuII showed higher in vitro affinity for OL than for OR, implicating the spacer sequences in this difference. Mutational analysis associated the repression with OR, which overlaps the promoter −35 hexamer but is otherwise dispensable for activation. A nonrepressing mutant exhibited poor establishment in new cells. Comparing promoter-operator regions from PvuII and 29 R-M systems controlled by C proteins revealed that the most-highly conserved sequence is the tetranucleotide spacer separating OL from OR. Any changes in that spacer reduced the stability of C.PvuII-operator complexes and abolished activation.
PMCID: PMC2175313  PMID: 17933763
24.  The active site of TthPolX is adapted to prevent 8-oxo-dGTP misincorporation 
Nucleic Acids Research  2013;42(1):534-543.
Full genome sequencing of bacterial genomes has revealed the presence of numerous genes encoding family X DNA polymerases. These enzymes play a variety of biological roles and, accordingly, display often striking functional differences. Here we report that the PolX from the heat-stable organism Thermus thermophilus (TthPolX) inserts the four dNTPs with strong asymmetry. We demonstrate that this behaviour is related to the presence of a single divergent residue in the active site of TthPolX. Mutation of this residue (Ser266) to asparagine, the residue present in most PolXs, had a strong effect on TthPolX polymerase activity, increasing and equilibrating the insertion efficiencies of the 4 dNTPs. Moreover, we show that this behaviour correlates with the ability of TthPolX to insert 8-oxo-dGMP. Although the wild-type enzyme inefficiently incorporates 8-oxo-dGMP, the substitution of Ser266 to asparagine resulted in a dramatic increase in 8-oxo-dGMP incorporation opposite dA. These results suggest that the presence of a serine at position 266 in TthPolX allows the enzyme to minimize the formation of dA:8-oxo-dGMP at the expense of decreasing the insertion rate of pyrimidines. We discuss the structural basis for these effects and the implications of this behaviour for the GO system (BER of 8-oxo-dG lesions).
PMCID: PMC3874185  PMID: 24084083
25.  Retrospective survey of chronic Q fever in Japan by using PCR to detect Coxiella burnetii DNA in paraffin-embedded clinical samples. 
Journal of Clinical Microbiology  1996;34(4):824-827.
We used PCR to detect Coxiella burnetii DNA in paraffin-embedded tissues obtained from patients with chronic endocarditis in which the etiological agent had been unknown. On the basis of the published nucleotide sequence of the C. burnetii htpB gene, primers were chosen to produce an amplified fragment of 285 bp. A total of 60 samples from 56 patients were tested for the presence of C. burnetii DNA. Five samples from four patients were found to be positive. All of the amplified DNA fragments possessed a TthHB8I restriction site, as predicted from the published sequence of C. burnetii. In one of the four positive patients, rickettsia-like particles were found in sections of tissue stained by Gimenez's method. This is the first report of chronic Q fever in Japan.
PMCID: PMC228900  PMID: 8815091

Results 1-25 (249939)