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

<p>Abstract</p> <p>Background</p> <p>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 <it>Thermus </it>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.</p> <p>Results</p> <p>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 <it>tspgwi </it>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 <it>Thermus </it>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 <it>Thermus </it>sp.) are dependent on the presence of divalent cations.</p> <p>Conclusion</p> <p>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.</p

DNA-FACE™ - An <em>Escherichia coli</em>-based DNA Amplification-Expression Technology for Automatic Assembly of Concatemeric ORFs and Proteins

DNA-FACE™ (DNA Fragment Amplification & Concatemeric Expressed Nucleic Acids and Proteins) is a universal biotechnological platform, developed as Escherichia coli (E. coli) system. It is based on the ordered, head-to-tail directional ligation of the amplified DNA fragments. The technology enables the construction of targeted biomolecules - genetically programmed, concatemeric DNA, RNA, and proteins, designed to fit a particular task. The constructed, “artificial” (never seen in Nature) tandem repeat macromolecules, with specialized functions, may contain up to 500 copies of monomeric units. The technology greatly exceeds the current capabilities of chemical gene synthesis. The vector-enzymatic DNA fragment amplification assembles the DNA segments, forming continuous Open Reading Frames (ORFs). The obtained ORFs are ready for high-level expression in E. coli without a need for subcloning. The presented method has potential applications in pharmaceutical industry and tissue engineering, including vaccines, biological drugs, drug delivery systems, mass-production of peptide-derived biomaterials, industrial and environmental processes. The technology has been patented worldwide and used successfully in the construction of anti-HBV vaccines, pro-regenerative biological drugs and, recently, the anti-SARS-CoV-2 vaccine. The anti-SARS-CoV-2 vaccine, developed using the DNA-FACE™ technology, is nontoxic and induces strong immunological response to recombinant human spike and nucleocapsid proteins, as shown in animal studies

Modified ‘one amino acid-one codon’ engineering of high GC content TaqII-coding gene from thermophilic Thermus aquaticus results in radical expression increase

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

Related bifunctional restriction endonuclease-methyltransferase triplets: TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities

<p>Abstract</p> <p>Background</p> <p>We previously defined a family of restriction endonucleases (REases) from <it>Thermus </it>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.</p> <p>Results</p> <p>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 <it>Thermus </it>sp. enzyme family, was cloned and characterized using a recently developed method for partially cleaving REases.</p> <p>Conclusions</p> <p>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.</p

A Method for Isolation Bacteriophage Particles-Free Genomic DNA, Exemplified by TP-84, Infecting Thermophilic Geobacillus

DNA purification methods are indispensable tools of molecular biology, used for many decades. Nevertheless, for certain specialized applications, the currently employed techniques are not sufficiently effective. While examining a number of the existing methods to purify the genomic DNA of the thermophilic bacteriophage TP-84, which infects Geobacillus stearothermophilus (G. stearothermophilus), we have found out that the obtained DNA is contaminated with trace amounts of infectious TP-84 particles. This was detrimental for the bacteriophage genetic manipulation purposes, as finding the recombinant TP-84 clones was essentially impossible due to the appearance of a high background of native bacteriophage plaques. Thus, we have developed a method, which enables the fast and efficient isolation of a bacteriophage genomic DNA from concentrated phage preparations, obtained using CsCl gradient ultracentrifugation, without the need to remove concentrated CsCl solutions. The method employs silica columns and mini-scale isolation of microgram amounts of high quality DNA. It is universal&mdash;the silica mini-columns from various manufacturers can be used to conduct the procedure. The purified DNA, free from infectious bacteriophage particles, is ready for further manipulations. This is particularly important for such thermophilic bacteriophages that may partially survive standard isolation procedures and contaminate the final DNA product

Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

Abstract Background Acoustic or hydrodynamic shearing, sonication and enzymatic digestion are used to fragment DNA. However, these methods have several disadvantages, such as DNA damage, difficulties in fragmentation control, irreproducibility and under-representation of some DNA segments. The DNA fragmentation tool would be a gentle enzymatic method, offering cleavage frequency high enough to eliminate DNA fragments distribution bias and allow for easy control of partial digests. Only three such frequently cleaving natural restriction endonucleases (REases) were discovered: CviJI, SetI and FaiI. Therefore, we have previously developed two artificial enzymatic specificities, cleaving DNA approximately every ~ 3-bp: TspGWI/sinefungin (SIN) and TaqII/SIN. Results In this paper we present the third developed specificity: TthHB27I/SIN(SAM) - a new genomic tool, based on Type IIS/IIC/IIG Thermus-family REases-methyltransferases (MTases). In the presence of dimethyl sulfoxide (DMSO) and S-adenosyl-L-methionine (SAM) or its analogue SIN, the 6-bp cognate TthHB27I recognition sequence 5’-CAARCA-3′ is converted into a combined 3.2–3.0-bp ‘site’ or its statistical equivalent, while a cleavage distance of 11/9 nt is retained. Protocols for various modes of limited DNA digestions were developed. Conclusions In the presence of DMSO and SAM or SIN, TthHB27I is transformed from rare 6-bp cutter to a very frequent one, approximately 3-bp. Thus, TthHB27I/SIN(SAM) comprises a new tool in the very low-represented segment of such prototype REases specificities. Moreover, this modified TthHB27I enzyme is uniquely suited for controlled DNA fragmentation, due to partial DNA cleavage, which is an inherent feature of the Thermus-family enzymes. Such tool can be used for quasi-random libraries generation as well as for other DNA manipulations, requiring high frequency cleavage and uniform distribution of cuts along DNA

Photoinduced Single Strand Breaks and Intrastrand Cross-Links in an Oligonucleotide Labeled with 5‑Bromouracil

5-Bromouracil (BrU) is photoreactive toward near UVB photons and can be introduced into genomic DNA during its biosynthesis in cells. However, PCR seems to be a simpler approach, which can be used to obtain labeled DNA similar to that synthesized within the cell. In the current work, PCR has been employed and optimized in order to substitute all thymines (besides those present in starters) with BrU in the dsDNA fragment of 80 base pairs (bp) in length. The modified oligonucleotide was irradiated with 300 nm photons in a buffered aqueous solution (pH = 7) and digested with a cocktail of enzymes specific to the phosphodiester bond cleavage. Initially, the extent of damage in the intact photolyte was measured with DHPLC. Then, the digested reaction mixture was subjected to HPLC and MS analyses and, in addition to the formation of 5-bromo-2′-deoxuyridine, which proves the occurrence of single strand breaks (SSBs) due to irradiation, U∧U and U∧C dimers were found, whose molecular structure was confirmed by MS/MS analysis. Although the abundance of such tandem lesions is lower than that of the SSB type, they pose a potent threat to genome integrity. Thus, our findings shed new light on the photosensitizing properties of BrU toward DNA

Comparison of RM.TthHB27I thermozyme production in native wt host <i>T</i>. <i>thermophilus</i> and in <i>E</i>. <i>coli</i> expressing both <i>tthHB27IRM</i> gene variants.

<p>Comparison of RM.TthHB27I thermozyme production in native wt host <i>T</i>. <i>thermophilus</i> and in <i>E</i>. <i>coli</i> expressing both <i>tthHB27IRM</i> gene variants.</p