Promising approaches for the assembly of the catalytically active, recombinant Desulfomicrobium baculatum hydrogenase with substitutions at the active site.

BACKGROUND: Hydrogenases (H2ases) are metalloenzymes capable of the reversible conversion of protons and electrons to molecular hydrogen. Exploiting the unique enzymatic activity of H2ases can lead to advancements in the process of biohydrogen evolution and green energy production. RESULTS: Here we created of a functional, optimized operon for rapid and robust production of recombinant [NiFe] Desulfomicrobium baculatum hydrogenase (Dmb H2ase). The conversion of the [NiFeSe] Dmb H2ase to [NiFe] type was performed on genetic level by site-directed mutagenesis. The native dmb operon includes two structural H2ase genes, coding for large and small subunits, and an additional gene, encoding a specific maturase (protease) that is essential for the proper maturation of the enzyme. Dmb, like all H2ases, needs intricate bio-production machinery to incorporate its crucial inorganic ligands and cofactors. Strictly anaerobic, sulfate reducer D. baculatum bacteria are distinct, in terms of their biology, from E. coli. Thus, we introduced a series of alterations within the native dmb genes. As a result, more than 100 elements, further compiled into 32 operon variants, were constructed. The initial requirement for a specific maturase was omitted by the artificial truncation of the large Dmb subunit. The assembly of the produced H2ase subunit variants was investigated both, in vitro and in vivo. This approach resulted in 4 recombinant [NiFe] Dmb enzyme variants, capable of H2 evolution. The aim of this study was to overcome the gene expression, protein biosynthesis, maturation and ligand loading bottlenecks for the easy, fast, and cost-effective delivery of recombinant [NiFe] H2ase, using a commonly available E. coli strains. CONCLUSION: The optimized genetic constructs together with the developed growth and purification procedures appear to be a promising platform for further studies toward fully-active and O2 tolerant, recombinant [NiFeSe] Dmb H2ase, resembling the native Dmb enzyme. It could likely be achieved by selective cysteine to selenocysteine substitution within the active site of the [NiFe] Dmb variant.

Modified Peptide Molecules As Potential Modulators of Shelterin Protein Functions; TRF1.

In this work, we present studies on relatively new and still not well-explored potential anticancer targets which are shelterin proteins, in particular the TRF1 protein can be blocked by in silico designed "peptidomimetic" molecules. TRF1 interacts directly with the TIN2 protein, and this protein-protein interaction is crucial for the proper functioning of telomere, which could be blocked by our novel modified peptide molecules. Our chemotherapeutic approach is based on assumption that modulation of TRF1-TIN2 interaction may be more harmful for cancer cells as cancer telomeres are more fragile than in normal cells. We have shown in vitro within SPR experiments that our modified peptide PEP1 molecule interacts with TRF1, presumably at the site originally occupied by the TIN2 protein. Disturbance of the shelterin complex by studied molecule may not in short term lead to cytotoxic effects, however blocking TRF1-TIN2 resulted in cellular senescence in cellular breast cancer lines used as a cancer model. Thus, our compounds appeared useful as starting model compounds for precise blockage of TRF proteins.

Data regarding a new, vector-enzymatic DNA fragment amplification-expression technology for the construction of artificial, concatemeric DNA, RNA and proteins, as well as biological effects of selected polypeptides obtained using this method.

Applications of bioactive peptides and polypeptides are emerging in areas such as drug development and drug delivery systems. These compounds are bioactive, biocompatible and represent a wide range of chemical properties, enabling further adjustments of obtained biomaterials. However, delivering large quantities of peptide derivatives is still challenging. Several methods have been developed for the production of concatemers - multiple copies of the desired protein segments. We have presented an efficient method for the production of peptides of desired length, expressed from concatemeric Open Reading Frame. The method employs specific amplification-expression DNA vectors. The main methodological approaches are described by Skowron et al., 2020 [1]. As an illustration of the demonstrated method's utility, an epitope from the S protein of Hepatitis B virus (HBV) was amplified. Additionally, peptides, showing potentially pro-regenerative properties, derived from the angiopoietin-related growth factor (AGF) were designed and amplified. Here we present a dataset including: (i) detailed protocols for the purification of HBV and AGF - derived polyepitopic protein concatemers, (ii) sequences of the designed primers, vectors and recombinant constructs, (iii) data on cytotoxicity, immunogenicity and stability of AGF-derived polypeptides.

A vector-enzymatic DNA fragment amplification-expression technology for construction of artificial, concatemeric DNA, RNA and proteins for novel biomaterials, biomedical and industrial applications.

A DNA fragment amplification/expression technology for the production of new generation biomaterials for scientific, industrial and biomedical applications is described. The technology enables the formation of artificial Open Reading Frames (ORFs) encoding concatemeric RNAs and proteins. It recruits the Type IIS SapI restriction endonuclease (REase) for an assembling of DNA fragments in an ordered head-to-tail-orientation. The technology employs a vector-enzymatic system, dedicated to the expression of newly formed, concatemeric ORFs from strong promoters. Four vector series were constructed to suit specialised needs. As a proof of concept, a model amplification of a 7-amino acid (aa) epitope from the S protein of HBV virus was performed, resulting in 500 copies of the epitope-coding DNA segment, consecutively linked and expressed in Escherichia coli (E. coli). Furthermore, a peptide with potential pro-regenerative properties (derived from an angiopoietin-related growth factor) was designed. Its aa sequence was back-translated, codon usage optimized and synthesized as a continuous ORF 10-mer. The 10-mer was cloned into the amplification vector, enabling the N-terminal fusion and multiplication of the encoded protein with MalE signal sequence. The obtained genes were expressed, and the proteins were purified. Conclusively, we show that the proteins are neither cytotoxic nor immunogenic and they have a very low allergic potential.

Novel parameter describing restriction endonucleases: Secondary-Cognate-Specificity and chemical stimulation of TsoI leading to substrate specificity change.

Over 470 prototype Type II restriction endonucleases (REases) are currently known. Most recognise specific DNA sequences 4-8 bp long, with very few exceptions cleaving DNA more frequently. TsoI is a thermostable Type IIC enzyme that recognises the DNA sequence TARCCA (R = A or G) and cleaves downstream at N11/N9. The enzyme exhibits extensive top-strand nicking of the supercoiled single-site DNA substrate. The second DNA strand of such substrate is specifically cleaved only in the presence of duplex oligonucleotides containing a cognate site. We have previously shown that some Type IIC/IIG/IIS enzymes from the Thermus-family exhibit 'affinity star' activity, which can be induced by the S-adenosyl-L-methionine (SAM) cofactor analogue-sinefungin (SIN). Here, we define a novel type of inherently built-in 'star' activity, exemplified by TsoI. The TsoI 'star' activity cannot be described under the definition of the classic 'star' activity as it is independent of the reaction conditions used and cannot be separated from the cognate specificity. Therefore, we define this phenomenon as Secondary-Cognate-Specificity (SCS). The TsoI SCS comprises several degenerated variants of the cognate site. Although the efficiency of TsoI SCS cleavage is lower in comparison to the cognate TsoI recognition sequence, it can be stimulated by S-adenosyl-L-cysteine (SAC). We present a new route for the chemical synthesis of SAC. The TsoI/SAC REase may serve as a novel tool for DNA manipulation.

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

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.

Sequence, genome organization, annotation and proteomics of the thermophilic, 47.7-kb Geobacillus stearothermophilus bacteriophage TP-84 and its classification in the new Tp84virus genus.

Bacteriophage TP-84 is a well-characterized bacteriophage of historical interest. It is a member of the Siphoviridae, and infects a number of thermophilic Geobacillus (Bacillus) stearothermophilus strains. Its' 47.7-kbp double-stranded DNA genome revealed the presence of 81 coding sequences (CDSs) coding for polypeptides of 4 kDa or larger. Interestingly, all CDSs are oriented in the same direction, pointing to a dominant transcription direction of one DNA strand. Based on a homology search, a hypothetical function could be assigned to 31 CDSs. No RNA or DNA polymerase-coding genes were found on the bacteriophage genome indicating that TP-84 relies on the host's transcriptional and replication enzymes. The TP84 genome is tightly packed with CDSs, typically spaced by several-to-tens of bp or often overlapping. The genome contains five putative promoter-like sequences showing similarity to the host promoter consensus sequence and allowing for a 2-bp mismatch. In addition, ten putative rho-independent terminators were detected. Because the genome sequence shows essentially no similarity to any previously characterised bacteriophage, TP-84 should be considered a new species in an undefined genus within the Siphoviridae family. Thus a taxonomic proposal of a new Tp84virus genus has been accepted by the International Committee on Taxonomy of Viruses. The bioinformatics genome analysis was verified by confirmation of 33 TP-84 proteins, which included: a) cloning of a selected CDS in Escherichia coli, coding for a DNA single-stranded binding protein (SSB; gene TP84_63), b) purification and functional assays of the recombinant TP-84 SSB, which has been shown to improve PCR reactions, c) mass spectrometric (MS) analysis of TP-84 bacteriophage capsid proteins, d) purification of TP-84 endolysin activity, e) MS analysis of the host cells from infection time course.

Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from Thermus thermophilus (RM.TthHB27I) cloned in Escherichia coli is critically affected by the codon composition of the synthetic gene.

Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes' expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified 'codon randomization' strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities.

A new prototype IIS/IIC/IIG endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus, recognising 5'-TARCCA(N11/9)-3' sequences.

The Thermus sp. family of IIS/IIG/IIC enzymes includes the thermostable, bifunctional, fused restriction endonuclease (REase)-methyltransferases (MTase): TaqII, Tth111II/TthHB27I, TspGWI, TspDTI and TsoI. The enzymes are large proteins (approximately 120kDa), their enzymatic activities are affected by S-adenosylmethionine (SAM), they recognise similar asymmetric cognate sites and cleave at a distance of 11/9 nucleotides (nt). The enzymes exhibit similarities of their amino acid (aa) sequences and DNA catalytic motifs. Thermus sp. enzymes are an example of functional aa sequence homologies among REases recognising different, yet related DNA sequences. The family consists of TspGWI- and TspDTI-subfamilies. TsoI appears to be a non-identical 'triplet', related to TspDTI and Tth111II/TthHB27I. The discovery of TsoI, purified from Thermus scotoductus, is described. This prototype, displaying a novel specificity, which was determined by: (i) cleavage of a reference plasmid and bacteriophage DNA, (ii) cleavage of custom PCR DNA substrates, (iii) run-off sequencing of cleavage products and (iv) shotgun cloning and sequencing of bacteriophage lambda (λ) DNA digested with TsoI. The enzyme recognises a degenerated 5'-TARCCA-3' sequence, whereas DNA strands are cut 11/9 nt downstream. The discovery of the TsoI prototype is of practical importance in biotechnology, as it extends the palette of cleavage specificities for gene cloning.

Two-stage gene assembly/cloning of a member of the TspDTI subfamily of bifunctional restriction endonucleases, TthHB27I.

The Thermus sp. family of bifunctional type IIS/IIG/IIC restriction endonucleases (REase)-methyltransferases (MTase) comprises thermo-stable TaqII, TspGWI, TspDTI, TsoI, Tth111II/TthHB27I enzymes as well as a number of putative enzymes/open reading frames (ORFs). All of the family members share properties including a large protein size (ca. 120kDa), amino acid (aa) sequence homologies, enzymatic activity modulation by S-adenosylmethionine (SAM), recognition of similar asymmetric cognate DNA sites and cleavage at a distance of 11/9 nt. Analysis of the enzyme aa sequences and domain/motif organisation led to further Thermus sp. family division into the TspDTI and TspGWI subfamilies. The latter exhibits an unprecedented phenomenon of DNA recognition change upon substitution of SAM by its analogue, sinefungin (SIN), towards a very frequent DNA cleavage. We report cloning in Escherichia coli (E. coli), using a two-stage procedure and a putative tthHB27IRM gene, detected by bioinformatics analysis of the Thermus thermophilus HB27 (T. thermophilus) genome. The functionality of a 3366 base pair (bp)-/1121 aa-long, high GC content ORF was validated experimentally through the expression in E. coli. Protein features corroborated with the reclassification of TthHB27I into the TspDTI subfamily, which manifested in terms of aa-sequence/motif homologies and insensitivity to SIN-induced specificity shift. However, both SAM and SIN stimulated the REase DNA cleavage activity by at least 16-32 times; the highest was observed for the Thermus sp. family. The availability of TthHB27I and the need to include SAM or SIN in the reaction in order to convert the enzyme from "hibernation" status to efficient DNA cleavage is of practical significance in molecular biotechnology, extending the palette of available REase specificities.

Artificial plasmid labeled with 5-bromo-2'-deoxyuridine: a universal molecular system for strand break detection.

DNA strand breaks (SBs) are among the most cytotoxic forms of DNA damage, and their residual levels correlate directly with cell death. Hence, the type and amount of SBs is directly related to the efficacy of a given anticancer therapy. In this study, we describe a molecular tool that can differentiate between single (SSBs) and double (DSBs) strand breaks and also assess them quantitatively. Our method involves PCR amplification of a linear DNA fragment labeled with a sensitizing nucleotide, circularization of that fragment, and enzymatic introduction of supercoils to transform the circular relaxed form of the synthesized plasmid into a supercoiled one. After exposure of the molecule to a damaging factor, SSB and DSB levels can be easily assayed with gel electrophoresis. We applied this method to prepare an artificial plasmid labeled with 5-bromo-2'-deoxyuridine and to assay SBs photoinduced in the synthesized plasmid.

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) PR 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.

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.

Three-stage biochemical selection: cloning of prototype class IIS/IIC/IIG restriction endonuclease-methyltransferase TsoI from the thermophile Thermus scotoductus.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

BACKGROUND: 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). RESULTS: 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. CONCLUSIONS: 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.

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

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.

Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence.

BACKGROUND: Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. RESULTS: An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb). CONCLUSIONS: The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy.

Chemically-induced affinity star restriction specificity: a novel TspGWI/sinefungin endonuclease with theoretical 3-bp cleavage frequency.

The type IIS/IIC restriction endonuclease TspGWI recognizes the sequence 5'-ACGGA-3', cleaving DNA 11/9 nucleotides downstream. Here we show that sinefungin, a cofactor analog of S-adenosyl methionine, induces a unique type of relaxation in DNA recognition specificity. In the presence of sinefungin, TspGWI recognizes and cleaves at least 12 degenerate variants of the original recognition sequence that vary by single base pair changes from the original 5-bp restriction site with only a single degeneracy per variant appearing to be allowed. In addition, sinefungin was found to have a stimulatory effect on cleavage at these nondegenerate TspGWI recognition sites, irrespective of their number or the DNA topology. Interestingly, no fixed "core" could be identified among the new recognition sequences. Theoretically, TspGWI cleaves DNA every 1024 bp, while sinefungin-induced activity cleaves every 78.8 bp, corresponding to a putative 3-bp long recognition site. Thus, the combination of sinefungin and TspGWI represents a novel frequent cutter, next only to CviJI/CviJI*, that should prove useful in DNA cloning methodologies.

Unexpected photoproduct generated via the acetone-sensitized photolysis of 5-bromo-2'-deoxyuridine in a water/isopropanol solution: experimental and computational studies.

The acetone-sensitized photolysis of 5-bromo-2'-deoxyuridine (5-BrdU) in a water/isopropanol solution with 300 nm photons leads to the formation of 2'-deoxyuridine (dU) and a comparable amount of another photoproduct that has not been reported in the literature so far. The negative and positive mass spectra recorded for this species indicate that they originate from the molecular mass of 286 Da, which corresponds to an adduct of 2'-deoxyuridine and 2-propanol. Quantum chemical calculations carried out at the DFT and TDDFT levels reveal both the structure and the UV spectrum of that adduct. The latter computational characteristic matches well the experimental UV spectrum of the new photoproduct. Our findings indicate that the acetone-sensitized photolysis of 5-BrdU is more complicated than has hitherto been assumed. Nevertheless, since electron transfer is one of the pathways responsible for 5-BrdU decay, acetone-sensitized photolysis of the halogen derivatives of nucleobases could be a convenient tool for studying their radiosensitivity in aqueous solutions.

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. 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.