Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family

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.     

Towards a better understanding of Lactobacillus rhamnosus GG - host interactions

Background

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).

Results

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.

Conclusions

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.     

A new Thermus sp. class-IIS enzyme sub-family: isolation of a 'twin' endonuclease TspDTI with a novel specificity 5'-ATGAA(N(11/9))-3', related to TspGWI,TaqII and Tth111II

The TspDTI restriction endonuclease, which shows a novel recognition specificity 5'-ATGAA(N(11/9))-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(N(11/9))-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.     

Cofactor analogue-induced chemical reactivation of endonuclease activity in a DNA cleavage/methylation deficient TspGWI N473A variant in the NPPY motif

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.     

A homology model of restriction endonuclease SfiI in complex with DNA

Background

Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified.

Results

SfiI is a REase which recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3' and generates 3 nt long 3' overhangs upon cleavage. SfiI is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between SfiI and BglI, a dimeric Type IIP restriction enzyme from the PD-DxK superfamily of nucleases, which recognizes the 5'GCCNNNN^NGGC3' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis.

Conclusions

The bioinformatics analysis suggest that SfiI, a Type IIF enzyme, is more closely related to BglI, an "orthodox" Type IIP restriction enzyme, than to any other REase, including other Type IIF REases with known structures, such as NgoMIV. NgoMIV and BglI belong to two different, very remotely related branches of the PD-DxK superfamily: the α-class (EcoRI-like), and the β-class (EcoRV-like), respectively. Thus, our analysis provides evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily of REases. The model of SfiI will also serve as a convenient platform for further experimental analyses.     

Type II restriction endonuclease R.Hpy188I belongs to the GIY-YIG nuclease superfamily,but exhibits an unusual active site

Background  

Catalytic domains of Type II restriction endonucleases (REases) belong to a few unrelated three-dimensional folds. While the PD-(D/E)XK fold is most common among these enzymes, crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI). Bioinformatics analyses supported by mutagenesis experiments suggested that some REases belong to the HNH fold (e.g. R.KpnI), and that a small group represented by R.Eco29kI belongs to the GIY-YIG fold. However, for a large fraction of REases with known sequences, the three-dimensional fold and the architecture of the active site remain unknown, mostly due to extreme sequence divergence that hampers detection of homology to enzymes with known folds.     

A putative Type IIS restriction endonuclease GeoICI from <Emphasis Type="BoldItalic">Geobacillus</Emphasis> sp. – A robust,thermostable alternative to mezophilic prototype BbvI

Screening of extreme environments in search for novel microorganisms may lead to the discovery of robust enzymes with either new substrate specificities or thermostable equivalents of those already found in mesophiles, better suited for biotechnology applications. Isolates from Iceland geysers’ biofilms, exposed to a broad range of temperatures, from ambient to close to water boiling point, were analysed for the presence of DNA-interacting proteins, including restriction endonucleases (REases). GeoICI, a member of atypical Type IIS REases, is the most thermostable isoschizomer of the prototype BbvI, recognizing/cleaving 5′-GCAGC(N_8/12)-3′ DNA sequences. As opposed to the unstable prototype, which cleaves DNA at 30°C, GeoICI is highly active at elevated temperatures, up to 73°C and over a very wide salt concentration range. Recognition/cleavage sites were determined by: (i) digestion of plasmid and bacteriophage lambda DNA (λ); (ii) cleavage of custom PCR substrates, (iii) run-off sequencing of GeoICI cleavage products and (iv) shotgun cloning and sequencing of λ DNA fragmented with GeoICI. Geobacillus sp. genomic DNA was PCR-screened for the presence of other specialized REases-methyltransferases (MTases) and as a result, another putative REase-MTase, GeoICII, related to the Thermus sp. family of bifunctional REases-MTases was detected.     

Exon sequence requirements for excision in vivo of the bacterial group II intron RmInt1

Background

Group II intron splicing proceeds through two sequential transesterification reactions in which the 5' and 3'-exons are joined together and the lariat intron is released. The intron-encoded protein (IEP) assists the splicing of the intron in vivo and remains bound to the excised intron lariat RNA in a ribonucleoprotein particle (RNP) that promotes intron mobility. Exon recognition occurs through base-pairing interactions between two guide sequences on the ribozyme domain dI known as EBS1 and EBS2 and two stretches of sequence known as IBS1 and IBS2 on the 5' exon, whereas the 3' exon is recognized through interaction with the sequence immediately upstream from EBS1 [(δ-δ' interaction (subgroup IIA)] or with a nucleotide [(EBS3-IBS3 interaction (subgroup IIB and IIC))] located in the coordination-loop of dI. The δ nucleotide is involved in base pairing with another intron residue (δ') in subgroup IIB introns and this interaction facilitates base pairing between the 5' exon and the intron.

Results

In this study, we investigated nucleotide requirements in the distal 5'- and 3' exon regions, EBS-IBS interactions and δ-δ' pairing for excision of the group IIB intron RmInt1 in vivo. We found that the EBS1-IBS1 interaction was required and sufficient for RmInt1 excision. In addition, we provide evidence for the occurrence of canonical δ-δ' pairing and its importance for the intron excision in vivo.

Conclusions

The excision in vivo of the RmInt1 intron is a favored process, with very few constraints for sequence recognition in both the 5' and 3'-exons. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting.     

Avipoxviruses: infection biology and their use as vaccine vectors

Background

It has been well documented that the 5' untranslated region (5' UTR) of many positive-stranded RNA viruses contain key cis-acting regulatory sequences, as well as high-order structural elements. Little is known for such regulatory elements controlling porcine arterivirus replication. We investigated the roles of a conserved stem-loop 2 (SL2) that resides in the 5'UTR of the genome of a type II porcine reproductive and respiratory syndrome virus (PRRSV).

Results

We provided genetic evidences demonstrating that 1) the SL2 in type II PRRSV 5' UTR, N-SL2, could be structurally and functionally substituted by its counterpart in type I PRRSV, E-SL2; 2) the functionality of N-SL2 was dependent upon the G-C rich stem structure, while the ternary-loop size was irrelevant to RNA synthesis; 3) serial deletions showed that the stem integrity of N-SL2 was crucial for subgenomic mRNA synthesis; and 4) when extensive base-pairs in the stem region was deleted, an alternative N-SL2-like structure with different sequence was utilized for virus replication.

Conclusion

Taken together, we concluded that the phylogenetically conserved SL2 in the 5' UTR was crucial for PRRSV virus replication, subgenomic mRNA synthesis in particular.     

Engineering TaqII bifunctional endonuclease DNA recognition fidelity: the effect of a single amino acid substitution within the methyltransferase catalytic site

The aim of this study was to improve a useful molecular tool—TaqII restriction endonuclease-methyltransferase—by rational protein engineering, as well as to show an application of our novel method of restriction endonuclease activity modulation through a single amino acid change in the NPPY motif of methyltransferase. An amino acid change was introduced using site-directed mutagenesis into the taqIIRM gene. The mutated gene was expressed in Escherichia coli. The protein variant was purified and characterized. Previously, we described a TspGWI variant with an amino acid change in the methyltransferase motif IV. Here, we investigate a complex, pleiotropic effect of an analogous amino acid change on its homologue—TaqII. The methyltransferase activity is reduced, but not abolished, while TaqII restriction endonuclease can be reactivated by sinefungin, with an increased DNA recognition fidelity. The general method for engineering of the IIS/IIC/IIG restriction endonuclease activity/fidelity is developed along with the generation of an improved TaqII enzyme for biotechnological applications. A successful application of our novel strategy for restriction endonuclease activity/fidelity alteration, based on bioinformatics analyses, mutagenesis and the use of cofactor-analogue activity modulation, is presented.     

Screening for restriction endonucleases in aerobic,thermophilic eubacteria

Summary A total of 216 Icelandic aerobic, heterotrophic, thermophiles belonging to three different genera were screened for type II restriction endonucleases. The frequency of positive strains was 44% for both Thermus and Bacillus but 63% for Rhodothermus. Approximately half of the enzymes from each group were characterised and a total of 14 different restriction enzymes were found. In all cases they were isoschizomers of known enzymes. Thermus contained 9 different types, Bacillus 6 and Rhodothermus had 3. This is the first time that isoschizomers of BspEI, BglI, EagI and EcoRV are found in Thermus and BstBI and EcoRV are found in Rhodothermus.     

Engineering better biomass-degrading ability into a GH11 xylanase using a directed evolution strategy

Background

Improving the hydrolytic performance of hemicellulases on lignocellulosic biomass is of considerable importance for second-generation biorefining. To address this problem, and also to gain greater understanding of structure-function relationships, especially related to xylanase action on complex biomass, we have implemented a combinatorial strategy to engineer the GH11 xylanase from Thermobacillus xylanilyticus (Tx-Xyn).

Results

Following in vitro enzyme evolution and screening on wheat straw, nine best-performing clones were identified, which display mutations at positions 3, 6, 27 and 111. All of these mutants showed increased hydrolytic activity on wheat straw, and solubilized arabinoxylans that were not modified by the parental enzyme. The most active mutants, S27T and Y111T, increased the solubilization of arabinoxylans from depleted wheat straw 2.3-fold and 2.1-fold, respectively, in comparison to the wild-type enzyme. In addition, five mutants, S27T, Y111H, Y111S, Y111T and S27T-Y111H increased total hemicellulose conversion of intact wheat straw from 16.7%_{tot. xyl} (wild-type Tx-Xyn) to 18.6% to 20.4%_{tot. xyl}. Also, all five mutant enzymes exhibited a better ability to act in synergy with a cellulase cocktail (Accellerase 1500), thus procuring increases in overall wheat straw hydrolysis.

Conclusions

Analysis of the results allows us to hypothesize that the increased hydrolytic ability of the mutants is linked to (i) improved ligand binding in a putative secondary binding site, (ii) the diminution of surface hydrophobicity, and/or (iii) the modification of thumb flexibility, induced by mutations at position 111. Nevertheless, the relatively modest improvements that were observed also underline the fact that enzyme engineering alone cannot overcome the limits imposed by the complex organization of the plant cell wall and the lignin barrier.     

A new aspect of a restriction endonuclease Tth111 I. It has a degenerated specificity (Tth111 I)

We previously reported that Thermus thermophilus 111 contained two restriction enzymes, Tth111 I and Tth111 II. The former does not cleave phi X174RFDNA and the latter does. We have now found another endonuclease activity able to cleave phi X174RFDNA in the cell extract of T. thermophilus 111. The protein with this activity was purified in a homogeneous state by chromatography on cellulose phosphate, heparin-Sepharose 4B and hydroxylapatite, successively. However, this endonuclease activity was always accompanied with Tth111 I activity during the purification procedure and the purified protein also showed a strong Tth111 I activity, suggesting that the Tth111 I activity and the phi X174RFDNA-cleaving activity reside in a single molecule. The phi X174RFDNA-cleaving activity was enhanced more strongly with Mn2+ than with Mg2+ and seemed to be attributable to a relaxed specificity of Tth111 I activity as seen in the cases of EcoRI* and BamHI* Thus we designated the phi X174RFDNA-cleaving activity Tth111 I*. The molecular weight of the protein with both Tth111 I and Tth111 I* activities was determined to be about 76,000 by gel filtration on a Sephadex G-100 column and 39,000 by SDS-polyacrylamide gel electrophoresis, suggesting the enzyme to be a dimer consisting of identical polypeptide chains. The phi X174RFDNA sequences surrounding Tth111 I* cuts were determined by the chain terminator method of Sanger et al. The results confirmed that Tth111 I* recognized a degenerated form of the Tth111 I recognition sequence, i.e., a sequence such that one of the specified nucleotides in the Tth111 I recognition sequence, 5'GACNNNGTC3', was substituted with N (N stands for any of A, G, C, and T), such as 5'NACNNNGTC3', 5'GACNNNNTC3', 5'GACNNNGNC, and so on (arrows indicate cleavage sites).     

The gene structure and expression of human ABHD1: overlapping polyadenylation signal sequence with Sec12

Background

Overlapping sense/antisense genes orientated in a tail-to-tail manner, often involving only the 3'UTRs, form the majority of gene pairs in mammalian genomes and can lead to the formation of double-stranded RNA that triggers the destruction of homologous mRNAs. Overlapping polyadenylation signal sequences have not been described previously.

Results

An instance of gene overlap has been found involving a shared single functional polyadenylation site. The genes involved are the human alpha/beta hydrolase domain containing gene 1 (ABHD1) and Sec12 genes. The nine exon human ABHD1 gene is located on chromosome 2p23.3 and encodes a 405-residue protein containing a catalytic triad analogous to that present in serine proteases. The Sec12 protein promotes efficient guanine nucleotide exchange on the Sar1 GTPase in the ER. Their sequences overlap for 42 bp in the 3'UTR in an antisense manner. Analysis by 3' RACE identified a single functional polyadenylation site, ATTAAA, within the 3'UTR of ABHD1 and a single polyadenylation signal, AATAAA, within the 3'UTR of Sec12. These polyadenylation signals overlap, sharing three bp. They are also conserved in mouse and rat. ABHD1 was expressed in all tissues and cells examined, but levels of ABHD1 varied greatly, being high in skeletal muscle and testis and low in spleen and fibroblasts.

Conclusions

Mammalian ABHD1 and Sec12 genes contain a conserved 42 bp overlap in their 3'UTR, and share a conserved TTTATTAAA/TTTAATAAA sequence that serves as a polyadenylation signal for both genes. No inverse correlation between the respective levels of ABHD1 and Sec12 RNA was found to indicate that any RNA interference occurred.     

Non-stem cancer cell kinetics modulate solid tumor progression

Background

Herpes simplex type II (HSV-2) is a member of the family herpesviridae. Human infection with this double stranded linear DNA virus causes genital ulcerative disease and existing treatment options only serve to resolve the symptomatology (ulcers) associated with active HSV-2 infection but do not eliminate latent virus. As a result, infection with HSV-2 follows a life-long relapsing (active versus latent) course. On the basis of a primitive bacterium anti-phage DNA defense, the restriction modification (R-M) system, we previously identified the Escherichia coli restriction enzyme (REase) EcoRII as a novel peptide to excise or irreversibly disrupt latent HSV-2 DNA from infected cells. However, sequences of the site specificity palindrome of EcoRII 5'-CCWGG-3' (W = A or T) are equally present within the human genome and are a potential source of host-genome toxicity. This feature has limited previous HSV-2 EcoRII based therapeutic models to microbicides only, and highlights the need to engineer artificial REases (zinc finger nucleases-ZFNs) with specificity to HSV-2 genomic-DNA only. Herein, the therapeutic-potential of zinc finger arrays (ZFAs) and ZFNs is identified and modeled, with unique specificity to the HSV-2 genome.

Methods and results

Using the whole genome of HSV-2 strain HG52 (Dolan A et al.,), and with the ZFN-consortium's CoDA-ZiFiT software pre-set at default, more than 28,000 ZFAs with specificity to HSV-2 DNA were identified. Using computational assembly (through in-silico linkage to the Flavobacterium okeanokoites endonuclease Fok I of the type IIS class), 684 ZFNs with specificity to the HSV-2 genome, were constructed. Graphic-analysis of the HSV-2 genome-cleavage pattern using the afore-identified ZFNs revealed that the highest cleavage-incidence occurred within the 30,950 base-pairs (~between the genomic context coordinates 0.80 and 1.00) at the 3' end of the HSV-2 genome. At approximately 3,095 bp before and after the 5' and 3' ends of the HSV-2 genome (genomic context coordinates 0.02 and 0.98, respectively) were specificity sites of ZFNs suited for the complete excision of over 60% of HSV-2 genomic material from within infected human cells, through the process of non-homologous end joining (NHEJ). Furthermore, a model concerning a recombinant (ICP10-PK mutant) replication competent HSV-2 viral vector for delivering and transducing a diploid copy (or pair) of the HSV-2-genome-specific ZFN genotype within neuronal tissue, is presented.

Conclusion

ZFNs with specificity to HSV-2 genomic DNA that are precursors of novel host-genome expressed HSV-2 gene-therapeutics or vaccines were identified.