Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving

<p>Abstract</p> <p>Background</p> <p>The TaqII enzyme is a member of the <it>Thermus </it>sp. enzyme family that we propounded previously within Type IIS restriction endonucleases, containing related thermophilic bifunctional endonucleases-methyltransferases from various <it>Thermus </it>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.</p> <p>Results</p> <p>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).</p> <p>Conclusions</p> <p>The DNA recognition sequence of the bifunctional prototype TaqII endonuclease-methyltransferase from <it>Thermus aquaticus </it>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.</p

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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).</p> <p>Conclusions</p> <p>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.</p

Construction of bionanoparticles with the use of a recombinant DNA vector-enzymatic system, containing artificial poliepitopic proteins, for the delivery of new generation vaccines

DNA/RNA amplification technologies, such as the Polymerase Chain Reaction have revolutionized modern biology, medical diagnostics and forensic analyses, among others. A number of alternative nucleic acids amplification methods have been developed, tailored to specific applications. Here we present a refined version of a DNA fragment amplification technology, which enables the construction of ordered concatemers in a head-to-tail-orientation. A very high number of DNA segments, at least 500 copies, can be consecutively linked. Other key features include: (i) the application of a dedicated vector-enzymatic system, including selected subtype IIS restriction endonucleases, which has been designed to automatically generate long Open Reading Frames and (ii) an amplification-expression vector with a built-in strong transcription promoter along with optimal translation initiation signals, which allow for a high level of expression of the constructed artificial poliepitopic protein. This highly advanced technology makes it possible to obtain ordered polymers of monomeric, synthetic or natural, DNA far beyond the capabilities of current chemical synthesis methods. The constructed poliepitopic proteins are further used for construction of several types of nanoparticles, including inclusion bodies and bacteriophages, containing multiple genetic fusion with poliepitopic proteins.The technology offers significant advances in a number of scientific, industrial and medical applications, including new vaccines and tissue pro-regenerative methods. The technology is protected by an international patent application and is available for licensing. Acknowledgments: project was supported by National Center for Research and Development, Warsaw, Poland, grant no STRATEGMED1/235077/9/NCBR/2014 and POIG.01.04.00-22-140/12; Jagiellonian Center for Innovation, Krakow, Poland; SATUS VC, Warsaw, Poland and BioVentures Institute Ltd, Poznan, Poland

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

A Method for Rapid Polyethyleneimine-Based Purification of Bacteriophage-Expressed Proteins from Diluted Crude Lysates, Exemplified by Thermostable TP-84 Depolymerase

Purification of bacteriophage-expressed proteins poses methodological difficulties associated with the need to process entire culture medium volume upon bacteriophage-induced bacterial cell lysis. We have used novel capsule glycosylase-depolymerase (TP84_26 GD) from bacteriophage TP-84, infecting thermophilic Geobacillus stearothermophilus bacteria, as a representative enzyme to develop a method for rapid concentration and purification of the enzyme present in diluted crude host cell lysate. A novel variant of the polyethyleneimine (PEI)-based purification method was devised that offers a fast and effective approach for handling PEI-facilitated purification of bacteriophage-expressed native proteins. Due to the very basic nature of PEI, the method is suitable for proteins interacting with nucleic acids or acidic proteins, where either mixed PEI-DNA or RNA–protein complexes or PEI–acidic protein complexes are reversibly precipitated. (i) The method is of general use, applicable with minor modifications to a variety of bacteriophage cell lysates and proteins. (ii) In the example application, TP84_26 GD was highly purified (over 50%) in a single PEI step; subsequent chromatography yielded a homogeneous enzyme. (iii) The enzyme’s properties were examined, revealing the presence of three distinct forms of the TP84_26 GD. These forms included soluble, unbound proteins found in host cell lysate, as well as an integrated form within the TP-84 virion