Characterization of a Family B DNA Polymerase from the Hyperthermophilic Crenarchaeon Ignicoccus hospitalis KIN4/I and Its Application to PCR

A family B DNA polymerase gene from the hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I was highly expressed under the control of T7lac promoter of pET-28ARG in Escherichia coli BL21-CodonPlus(DE3)-RIL cells. The produced I. hospitalis (Iho) DNA polymerase was purified by heat treatment followed by HisTrap? HP column and HiTrap? SP column chromatographies. The molecular mass of the purified Iho DNA polymerase was 88 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH for Iho DNA polymerase activity was 7.0 and the optimal temperature was 70 °C. Iho DNA polymerase was strongly activated by the presence of magnesium ion at an optimum concentration of 3 mM. The optimal concentration of KCl for Iho DNA polymerase activity was 60 mM. The half-life of the enzyme at 94 °C was about 2 h. The optimal conditions for polymerase chain reaction (PCR) were determined. Iho DNA polymerase possesses 3′?→?5′ exonuclease activity, and the fidelity of the Iho DNA polymerase was similar to that of Pfu and Vent DNA polymerases. However, Iho DNA polymerase provided more enhanced efficiency of PCR amplification than Pfu and Vent DNA polymerases. Iho DNA polymerase could successfully amplify a 2-kb λ DNA target with a 10-s extension time and could amplify a DNA fragment up to 8 kb λ DNA.     

Chemical properties of fatty acid derivatives as inhibitors of DNA polymerases

In this study, the chemical properties of organic acids as DNA polymerase inhibitors were examined. In total, we assayed the inhibitory activities of 23 compounds. We found that the DNA synthesis activity of DNA polymerase was usually reduced to less than 50% in the presence of 100 microM monoprotic acids, which have a Clog P value greater than 7.0 and a pK(a) value less than 5.4. With a minor modification these chemical properties applied to several organic fatty acids previously reported as DNA polymerase inhibitors. Moreover, we also examined the inhibitory activities of perfluorooctadecanoic acid (PFOdA) and perfluorooctanesulfonic acid (PFOS) against DNA polymerase beta in detail. These compounds inhibited the polymerase activity of pol beta competitively with template-primer DNA, and non-competitively with dNTPs. In addition, the 8 kDa domain-defective pol beta was also sensitive to these compounds. Our results suggest that the inhibitory mode of action of PFOdA and PFOS is different from that mediated by the classic fatty acid inhibitors against DNA polymerase beta.     

In vitro replication study of modified bases in ras sequences.

DNA templates containing a modified base (O6-methylguanine, 8-hydroxyguanine, xanthine or hypoxanthine) which was located in nucleotide sequences corresponding to the 12th or 61st codon of a ras gene were synthesized and deoxynucleotide incorporation opposite the lesions was investigated. The templates were replicated by Taq DNA polymerase, recombinant rat DNA polymerase beta and mouse DNA polymerase alpha-primase complex. Sequence analysis of the replicated products indicated selective incorporation of nucleotide(s) opposite a modified base, depending on the kind of base and of DNA polymerase. This system is very useful to obtain results of in vitro replication of modified bases in ras sequences.     

Biochemical Characterization of Translesion Synthesis by Sulfolobus acidocaldarius DNA Polymerases

To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarchaeota, two DNA polymerases of B family(polB1 and polB3), and one DNA polymerase of Y family(polIV) were recombinantly expressed, purified and biochemically characterized. Both DNA polymerases polB1(Saci_1537) and polB3(Saci_0074) possessed DNA polymerase and 3' to 5' exonuclease activities; however, both the activities of B3 were very inefficient in vitro. The polIV(Saci_0554) was a polymerase, not an exonuclease. The activities of all the three DNA polymerases were dependent on divalent metal ions Mn²⁺ and Mg²⁺. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KCl with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70℃. Deaminated bases dU and dI on DNA template strongly hindered primer extension by the two DNA polymerases of B family, not by the DNA polymerase of Y family. DNA polymerase of Y Family bypassed the two AP site analogues dSpacer and propane on template more easily than DNA polymerases of B family. Our results suggest that the three DNA polymerases coordinate to fulfill various DNA synthesis in Sulfolobus acidocaldarius cell.     

KF polymerase-based fluorescence aptasensor for the label-free adenosine detection

We have developed a simple, inexpensive, and label-free method for the selective detection of adenosine. Klenow fragment polymerase (KF polymerase) is a commonly-used 5' to 3' DNA polymerase, it also has 3' to 5' exonuclease activity that can digest single-stranded DNA. An adenosine binding DNA aptamer was employed, the aptamer was split into two pieces of single-stranded DNA (aptamer-A1 + aptamer-A2). Without the addition of adenosine, aptamer-A1 and aptamer-A2 existed as single-stranded DNA which could be efficiently degraded by the exonuclease activity of KF polymerase. Much reduced background fluorescence was obtained when SYBR Green dye was added. However, in the presence of adenosine, aptamer-A1 and aptamer-A2 bound to adenosine, and hybridization of the complementary sequences resulted in the formation of a duplex DNA structure, which could initiate DNA polymerization. The addition of SYBR Green dye resulted in a very high fluorescence enhancement, which could be used for the quantification of adenosine.     

White spot syndrome virus Orf514 encodes a bona fide DNA polymerase

White spot syndrome virus (WSSV) is the causative agent of white spot syndrome, one of the most devastating diseases in shrimp aquaculture. The genome of WSSV includes a gene that encodes a putative family B DNA polymerase (ORF514), which is 16% identical in amino acid sequence to the Herpes virus 1 DNA polymerase. The aim of this work was to demonstrate the activity of the WSSV ORF514-encoded protein as a DNA polymerase and hence a putative antiviral target. A 3.5 kbp fragment encoding the conserved polymerase and exonuclease domains of ORF514 was overexpressed in bacteria. The recombinant protein showed polymerase activity but with very low level of processivity. Molecular modeling of the catalytic protein core encoded in ORF514 revealed a canonical polymerase fold. Amino acid sequence alignments of ORF514 indicate the presence of a putative PIP box, suggesting that the encoded putative DNA polymerase may use a host processivity factor for optimal activity. We postulate that WSSV ORF514 encodes a bona fide DNA polymerase that requires accessory proteins for activity and maybe target for drugs or compounds that inhibit viral DNA replication.     

Replacing 32 proline residues by a noncanonical amino acid results in a highly active DNA polymerase

Protein engineering may be achieved by rational design, directed evolution-based methods, or computational protein design. Mostly these methods make recourse to the restricted pool of the 20 natural amino acids. With the ability to introduce different new kinds of functionalities into proteins, the use of noncanonical amino acids became a promising new method in protein engineering. Here, we report on the generation of a multifluorinated DNA polymerase. DNA polymerases are highly dynamic enzymes that catalyze DNA synthesis in a template-dependent manner, thereby passing several conformational states during the catalytic cycle. Here, we globally replaced 32 proline residues by the noncanonical imino acid (4R)-fluoroproline in a DNA polymerase of 540 amino acids (KlenTaq DNA polymerase). Interestingly, the substitution level of the proline residues was very efficient (92%). Nonetheless, the introduction of (4R)-fluoroproline into the DNA polymerase resulted in a highly active fluorinated enzyme, which was investigated in primer extension and PCR assays to analyze activity, selectivity, and stability in comparison to the parental enzyme. The DNA polymerase retained fidelity, activity, and sensitivity as the parental wild-type enzyme accompanied by some loss in thermostability. These results demonstrate that a noncanonical amino acid can be used for substitutions of natural counterparts in a highly dynamic enzyme with high molecular weight without effecting crucial enzyme properties. Furthermore, the employed DNA polymerase represents a promising starting point for directed DNA polymerase evolution with noncanonical amino acids.     

A facile graphene oxide-based DNA polymerase assay

The DNA polymerase assay is fundamental for related molecular biology investigations and drug screenings, however, the commonly used radioactive method is laborious and restricted. Herein, we report a novel, simple and cost-effective fluorometric DNA polymerase detection method by utilizing graphene oxide (GO) as a signal switch. In this strategy, in the absence of DNA polymerase, the fluorophore-labeled template ssDNA could be strongly adsorbed and almost entirely quenched by GO. However, as DNA polymerase exists, the polymerized dsDNA product might lead to a much lower quenching efficiency after addition of GO due to the much weaker interaction of dsDNA with GO than ssDNA, thus resulting in a much higher fluorescence signal detected. As proof of concept, the quantitative DNA polymerase activity assay was performed using the Klenow fragment exo(-) (KF(-)) as a model. It was confirmed that, after optimization of detection conditions, KF(-) activity could be sensitively detected through facile fluorescence measurements, with a detection limit of 0.05 U mL(-1) and a good linear correlation between 0.05-2.5 U mL(-1) (R(2) = 0.9928). In addition, this GO-based method was further inspected to evaluate the inhibitive behaviors of several drugs toward KF(-) activity, the result of which firmly demonstrated its potential application in polymerization-targeted drug screening.     

Primer Extension Reaction Assays for Incorporation of Deoxynucleotide Analogue into DNA

Incorporation of deoxynucleotide analogues into DNA is important for the expansion of DNA functions. Primer extension reactions are commonly used for the assay of such reaction events. However, current assay protocols generally rely on radiolabeling, fluorescence reporter labeling, or removal of specific deoxynucleotide triphosphate in the reaction mixture. Herein we report on the design of two novel assay protocols that utilize a dideoxynucleotide‐terminated template strand and a phosphorothiolate‐modified deoxynucleotide‐terminated template strand. We designed and synthesized a deoxyuridine triphosphate analogue (dU*TP) containing 2‐bromoisobutyryl group and demonstrated that it could be well recognized by ?29DNA polymerase, E. coli DNA polymerase I Klenow Fragment, Bst DNA polymerase Large Fragment, and E. coli DNA polymerase I Klenow Fragment (exo⁽), which translated to effective incorporation of dU*TP into DNA. dU*TP was also successfully incorporated into extremely long single‐stranded DNA at high‐density using ?29 DNA polymerase by rolling circle amplification.     

Highly efficient modification of DNA polymerase β under conditions of direct and sensitized activation of photoreactive DNAs. Modification of cell extract proteins

dUTP and dCTP derivatives containing a 4-azido-2,3,5,6-tetrafluorobenzylideneaminooxy group were incorporated into the 3′-end of the DNA primer within complexes with the DNA-matrix as analogs of natural dTTP by virtue of catalytic activity of DNA polymerase β or endogenous DNA polymerases of the cell extract. The photoreactive DNAs synthesized in situ were used for affinity modification of DNA polymerase β and DNA-binding proteins of the cell extract. For the photoreactive DNA based on these analogs, the efficiency of formation of covalent adducts with DNA polymerase β under the highest degree of DNA complexation with the enzyme was determined. The yield of covalent DNA adducts with the enzyme was 28–47%, depending on the type of the analog. The effect of the sequence of the DNA template near the localization of the photoreactive group on the redistribution of covalent cross-links between the possible targets was demonstrated. A possibility of increasing the efficiency of DNA polymerase β modification in the presence of a substantial excess of photoreactive DNA using a sensitizer, a dUTP derivative containing a pyrene residue, was studied. When photoreactive DNA containing a 2,3,5,6-tetrafluoro-4-azidobenzoyl (FAB) group was used, about 60% of DNA polymerase β was covalently attached to DNA. Photoreactive dNTP analogs ensuring a high level of protein modification in the cell extract were found. __________ Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1273–1283, May, 2005.     

Template-Dependent Incorporation of Spin-Labeled Thymidine Analogs into Viral DNA

The synthesis of novel pppU_d analogs substituted at position C(5) with tethered nitroxide radicals is reported. It is shown that these analogs can serve as good substrates for E. coli DNA polymerase (Pol I) and T-4 DNA polymerase using lambda-phage DNA as template. The template-dependent incorporation of the substrates was established by radio-labeling and ESR experiments.     

Impact and mechanism of TiO_2 nanoparticles on DNA synthesis in vitro

The impact of TiO2 nanoparticles on DNA synthesis in vitro in the dark and the molecular mechanism of such impact were studied. The impact of TiO2 nanoparticles on DNA synthesis was investigated by adding TiO2 nanoparticles in different sizes and at various concentrations into the polymerase chain reaction (PCR) system. TiO2 nanoparticles were premixed with the DNA polymerase, the primer or the template, respectively and then the supernatant and the precipitation of each mixture were added into the PCR system separately to observe the impact on DNA synthesis. Sequentially the interaction be- tween TiO2 nanoparticles and the DNA polymerase, the primer or the template was further analyzed by using UV-visible spectroscopy and polyacrylamide gel electrophoresis (PAGE). The results suggest that TiO2 nanoparticles inhibit DNA synthesis in the PCR system in the dark more severely than mi- croscale TiO2 particles at the equivalent concentration and the inhibition effect of TiO2 nanoparticles is concentration dependent. The molecular mechanism of such inhibition is that in the dark, TiO2 nanoparticles interact with the DNA polymerase through physical adsorption while TiO2 nanoparticles do with the primer or the template in a chemical adsorption manner. The disfunction levels of the bio-molecules under the impact of TiO2 nanoparticles are in the following order: the primer > the tem- plate > the DNA polymerase.     

Effects of DNA mismatches on binding affinity and kinetics of polymerase-DNA complexes as revealed by surface plasmon resonance biosensor

In this study, surface plasmon resonance (SPR) biosensor techniques were used to obtain quantitative information on the kinetics of the DNA and polymerase I (Klenow fragment) interaction. DNA duplexes containing different base compositions at the binding site were immobilized on the SPR sensor surface via biotin-streptavidin chemistry and the subsequent binding of the polymerase was measured in real time. Various kinetic models were tested and a translocation model was shown to provide the best fit for the binding and dissociation profiles. The results revealed that the enzyme binds to DNA at both the polymerase and the exonuclease domains with different association and dissociation rates as well as affinity constants, depending on the presence of mismatches near the primer 3'-end. Introduction of unpaired bases increases the DNA binding affinity towards the exonuclease domain and promotes the translocation of DNA from the polymerase site to the exonuclease site. The results also demonstrated that SPR biosensors may be used as a sensitive technique for studying molecular recognition events such as single-base discrimination involved in protein-DNA interaction.     

Direct Sensing of 5‐Methylcytosine by Polymerase Chain Reaction

The epigenetic control of genes by the methylation of cytosine resulting in 5‐methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′‐mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation‐specific PCR approach directly from untreated human genomic DNA.     

THYMIDINE UPTAKE, INCORPORATION AND DNA POLYMERASE ACTIVITY IN MURINE BLADDER TUMOR CELL,MBT–2, EXPOSED TO UV ACTIVATED DIHEMATOPORPHYRIN ETHER

Abstract— Photodynamic therapy (PDT) is a new modality for treatment of malignancy. In this paper, we reported the effect of UV activated dihematoporphyrin ether (DHE) on [³H] thymidine uptake and DNA synthesis in murine bladder tumor cells,MBT–2. Exponentially growing cells were pretreated with 0.05–5 μg/ml of DHE for 30 min in complete darkness prior to irradiation with 0.15-0.90 J/cm² of UV light (265 nm). The rates of thymidine uptake and DNA synthesis were suppressed in a DHE concentration and photic energy dependent manner. Double reciprocal analysis on the kinetics of the thymidine uptake and DNA synthesis indicated that the inhibition was non-competitive, i.e. decrease in both the apparent K^m value and maximum velocity in DHE plus UV light treated cells. The activities of DNA polymerase a and (3 were determined by [*H] dATP incorporation into DNA of permeabilizedMBT–2 cells. DNA polymerase a activity was approximately 60% of the control after 0.45 J/cm² of UV light exposure; a further inhibition of DNA polymerase a was observed when 0.5–5ng/W of DHE and UV photoradiation were combined. In contrast, a slight stimulation of DNA polymerase fJ was noted after a similar treatment. This study demonstrates that photodynamic therapy-induced suppression of DNA synthesis inMBT–2 cells is a complex process involving in reduction of thymidine transport as well as the perturbation of the enzymes involved in DNA synthesis.     

The 8-oxo-dGTP interaction with human DNA polymerase β: two patterns of ligand behavior

The behavior of 8-oxo-dGTP molecule in the area of the active site of human DNA polymerase β was investigated using molecular dynamics (MD) calculation. The principle phenomenon revealed as investigation results is existence of two cardinally different models of behavior inherent in 8-oxo-dGTP molecule. In the several cases, 8-oxo-dGTP stably stays in DNA polymerase active site, “keeps in touch” with template nucleotide and maintains the hydrogen bonds with it (stable behavior). In other cases, the ligand molecules lose the connections with template dA and start to migrate inside of enzyme space (migrate behavior). The 8-oxo-dGTP in cases of migrate behavior is still in DNA polymerase space at least over 100 ns and prevents transit of DNA polymerase from closed to open conformation as well as the further binding of incoming dNTP. This observation lets a possibility to consider it as natural inhibitor/regulator of DNA pol β activity.     

Exonuclease activity of proofreading DNA polymerases is at the origin of artifacts in molecular profiling studies

CE fingerprint methods are commonly used in microbial ecology. We have previously noticed that the position and number of peaks in CE-SSCP (single-strand conformation polymorphism) profiles depend on the DNA polymerase used in PCR [1]. Here, we studied the fragments produced by Taq polymerase as well as four commercially available proofreading polymerases, using the V3 region of the Escherichia coli rss gene as a marker. PCR products rendered multiple peaks in denaturing CE; Taq polymerase was observed to produce the longest fragments. Incubation of the fragments with T4 DNA polymerase indicated that the 3'-ends of the proofreading polymerase amplicons were recessed, while the Taq amplicon was partially +A tailed. Treatment of the PCR product with proofreading DNA polymerase rendered trimmed fragments. This was due to the 3'-5' exonuclease activity of these enzymes, which is essential for proofreading. The nuclease activity was reduced by increasing the concentration of dNTP. The Platinum Pfx DNA polymerase generated very few artifacts and could produce 85% of blunted PCR products. Nevertheless, despite the higher error rate, we recommend the use of Taq polymerase rather than proofreading in the framework for molecular fingerprint studies. They are more cost-effective and therefore ideally suited for high-throughput analysis; the +A tail artifact rate can be controlled by modifying the PCR primers and the reaction conditions.     

Effects of photo-activated bleomycin on deoxyribonuclease I, exonuclease III and deoxyribonucleic acid polymerase I reactions

The activity of bleomycin to break the strand of deoxyribonucleic acid (DNA) in the presence of 2-hydroxy-1-ethanethiol (2-mercaptoethanol) was enhanced by ultraviolet (UV) irradiation. Photo-activated bleomycin stimulated the action of deoxyribonuclease I (DNase I) to degrade DNA and the DNA synthesis by DNA polymerase I with DNase I. On the other hand, although UV-irradiated bleomycin scarcely broke the DNA strand in the presence of 1,2-benzenediol (catechol), it stimulated the action of DNase I to degrade DNA in the presence of catechol. In accordance with the inhibition by catechol, when DNA treated with UV-irradiated bleomycin in the presence of catechol was employed as a primer for the DNA synthesis, the incorporation of precursor into the acid-insoluble fraction by DNA polymerase I with exonuclease III was reduced to about one-half of the incorporation into DNA treated with unirradiated bleomycin. These findings suggest that the ability of bleomycin to bind to double-helical DNA forming regions sensitive to DNase I was increased by an appropriate dose of UV irradiation and that catechol inhibited the activity of the UV-irradiated bleomycin to break the DNA strand rather than to bind to DNA.     

Advantages of Thermococcus kodakaraenis (KOD) DNA Polymerase for PCR-mass spectrometry based analyses

The advantages of the thermostable DNA polymerase from Thermococcus kodakaraensis (KOD) are demonstrated for PCR amplification with subsequent detection by mass spectrometry. Commonly used DNA polymerases for PCR amplification include those from Thermus aquaticus (Taq) and Pyrococcus furiosus (Pfu). A 116 base-pair PCR product derived from a vWA locus was amplified by Taq, Pfu, or KOD DNA polymerase and compared by agarose gel electrophoresis and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). KOD DNA polymerase demonstrated a 2- to 3-fold increase in PCR product formation compared to Pfu or Taq, respectively, and generated blunt-ended PCR product which allows facile interpretation of the mass spectrum. Additionally, we demonstrate the advantage of using high magnetic fields to obtain unit resolution of the same 116 base pair (approximately 72 kDa) PCR product at high m/z.