Inhibition Of DNA Polymerase Activity Research Articles

Two-Metal-Ion Catalysis: Inhibition of DNA Polymerase Activity by a Third Divalent Metal Ion.

Almost all DNA polymerases (pols) exhibit bell-shaped activity curves as a function of both pH and Mg2+ concentration. The pol activity is reduced when the pH deviates from the optimal value. When the pH is too low the concentration of a deprotonated general base (namely, the attacking 3′-hydroxyl of the 3′ terminal residue of the primer strand) is reduced exponentially. When the pH is too high the concentration of a protonated general acid (i.e., the leaving pyrophosphate group) is reduced. Similarly, the pol activity also decreases when the concentration of the divalent metal ions deviates from its optimal value: when it is too low, the binding of the two catalytic divalent metal ions required for the full activity is incomplete, and when it is too high a third divalent metal ion binds to pyrophosphate, keeping it in the replication complex longer and serving as a substrate for pyrophosphorylysis within the complex. Currently, there is a controversy about the role of the third metal ion which we will address in this review.

Titanium Tackles the Endoplasmic Reticulum: A First Genomic Study on a Titanium Anticancer Metallodrug.

SummaryPhenolaTi is an advanced non-toxic anticancer chemotherapy; this inert bis(phenolato)bis(alkoxo) Ti(IV) complex demonstrates the intriguing combination of high and wide efficacy with no detected toxicity in animals. Here we unravel the cellular pathways involved in its mechanism of action by a first genome study on Ti(IV)-treated cells, using an attuned RNA sequencing-based available technology. First, phenolaTi induced apoptosis and cell-cycle arrest at the G2/M phase in MCF7 cells. Second, the transcriptome of the treated cells was analyzed, identifying alterations in pathways relating to protein translation, DNA damage, and mitochondrial eruption. Unlike for common metallodrugs, electrophoresis assay showed no inhibition of DNA polymerase activity. Reduced in vitro cytotoxicity with added endoplasmic reticulum (ER) stress inhibitor supported the ER as a putative cellular target. Altogether, this paper reveals a distinct ER-related mechanism by the Ti(IV) anticancer coordination complex, paving the way for wider applicability of related techniques in mechanistic analyses of metallodrugs.

Highly sensitive real-time PCR method to identify species origin in heparinoids.

Heparinoids are the starting material for sulodexide production, a drug used as intravenous anti-coagulant, as an alternative to heparin. The origin determination in the starting material for sulodexide, heparin, and derivatives is crucial for safety (including the impact related to bovine spongiform encephalopathy) and efficacy of the final products. Therefore, European countries have decided to approve the production of heparin only from porcine intestinal mucosa. PCR (polymerase chain reaction) methods are available to evaluate the origin species of crude heparin, during heparin production process, while they lack for the same analysis in heparinoids during sulodexide manufacturing processes. Notably, two main critical issues occur during the origin determination by using PCR for heparinoid analysis: first, heparin has been known to inhibit DNA polymerase activity and, second, the DNA amounts are very low in these samples. To overcome these critical issues, our proposed method is based on two fundamental steps, the DNA concentration by glycogen treatment and DNA purification, which occur before and after DNA extraction, respectively. Finally, by applying real-time PCR, we amplify three specific DNA sequences of ruminant species (bovine, ovine, and caprine), to assess possible contamination, and one from swine, to confirm the origin species. To date, such a method is the only one that determines origin species by PCR for heparinoids that guarantee quality, safety, and traceability of heparin-derived pharmaceutical products. In conclusion, our proposed method is an alternative to nuclear magnetic resonance and ELISA methods, because real-time PCR offers significant advantages in sensitivity, specificity, and robustness. Graphical Abstract.

Sensitive and specific detection of proteins based on target-responsive DNA polymerase activity

We herein describe a novel method for the detection of target protein based on the target-responsive DNA polymerase activity. In the sensor, two different types of DNA aptamers with the respective functions: one binds to the target protein and the other binds to DNA polymerase, are rationally engineered and combined to form the detection probe that regulates DNA polymerase activity in response to the target protein. In the presence of target protein, the detection probe becomes destabilized and stops the inhibition of DNA polymerase activity. Consequently, the active DNA polymerase initiates the primer extension reaction on the target-specific DNA aptamer, which recycles the target protein to promote another activation cycle of DNA polymerase. In addition, DNA polymerase also catalyzes the primer extension reaction on the primer/template complex in conjugation with TaqMan probe, leading to the significantly enhanced fluorescence intensities. With this novel strategy, we detected a model target protein, lysozyme with a limit of detection as low as 0.80 nM. In addition, the practical applicability of this system was successfully demonstrated by determining lysozyme in human serum.

Evaluating the effects of anticoagulants on Rhodnius prolixus artificial blood feeding.

Blood-sucking insects are responsible for the transmission of several important disease-causing organisms such as viruses, bacteria, and protozoans. The hematophagous hemipteran Rhodnius prolixus is one of the most important vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. Due to the medical importance of this insect, it has been used as a study model in physiology and biochemistry since the 1930s. Artificial feeding has been recognized as a feasible and a more ethical alternative method of feeding these hematophagous insects. To prevent clotting after blood collection defibrination or treatment with anticoagulants are necessary. Although anticoagulants have been routinely used for stabilizing the collected blood, there is a gap in demonstration of the effects of using anticoagulants on the feeding and development of the hematophagous insect Rhodnius prolixus. In this study, we compared the survival rate, molting efficiency, fertility, and infection development between insects that were fed on blood containing three different anticoagulants (citrate, EDTA, and heparin). We observed that fifth instar nymphs that were fed on blood containing EDTA and citrate could not perform digestion properly, which resulted in molting inefficiency. Adult insects that were fed on EDTA-containing blood laid lower number of eggs, and also had a diminished egg hatch percentage. When we delivered T. cruzi parasites in blood containing citrate or EDTA to the insects, a lower number of parasites and metacyclic trypomastigotes was observed in the intestine compared to the group fed on heparin-containing blood. Since heparin could potentially inhibit DNA polymerase activity in DNA samples extracted from the intestine, we analyzed different heparin concentrations to determine which one is the best for use as an anticoagulant. Concentrations ranging between 2.5 and 5 U/mL were able to inhibit coagulation without severely impairing DNA polymerase activity, thus indicating that this should be considered as the range of use for feeding experiments. Our results suggest that among the three anticoagulants tested, heparin can be recommended as the anticoagulant of choice for R. prolixus feeding experiments.

Inhibiting DNA Polymerases as a Therapeutic Intervention against Cancer.

Inhibiting DNA synthesis is an important therapeutic strategy that is widely used to treat a number of hyperproliferative diseases including viral infections, autoimmune disorders, and cancer. This chapter describes two major categories of therapeutic agents used to inhibit DNA synthesis. The first category includes purine and pyrmidine nucleoside analogs that directly inhibit DNA polymerase activity. The second category includes DNA damaging agents including cisplatin and chlorambucil that modify the composition and structure of the nucleic acid substrate to indirectly inhibit DNA synthesis. Special emphasis is placed on describing the molecular mechanisms of these inhibitory effects against chromosomal and mitochondrial DNA polymerases. Discussions are also provided on the mechanisms associated with resistance to these therapeutic agents. A primary focus is toward understanding the roles of specialized DNA polymerases that by-pass DNA lesions produced by DNA damaging agents. Finally, a section is provided that describes emerging areas in developing new therapeutic strategies targeting specialized DNA polymerases.

A Simple, Efficient and Rapid Method for Good Quality DNA Extraction from Rice Grains

An efficient and good DNA extraction protocol should be simple, affordable and yield enough DNA with high quality. Rice (Oryza sativa L.) DNA extraction methods often use seedlings or leaves rather than the grains and tend to be time-consuming, involve multiple steps, and use hazardous chemicals and expensive enzymes. Rice grains offer several benefits over seedlings and leaves as a source of DNA for genetic analysis. However, these benefits are underutilized because the bulk of a rice grain is made up of starch. It is particularly important, but difficult to get rid of the starch while extracting DNA from rice grains. This co-precipitated polysaccharide is a known inhibitor of DNA polymerase activity in polymerase chain reaction (PCR). We describe here a very simple and highly affordable Chelex®-100 based DNA extraction method from rice grains. It does not require any hazardous chemicals or enzymes. This method reproducibly extracts DNA with good purity indices (A260/A230 and A260/A280 values), but requires only a few steps.

Honokiol Inhibits DNA Polymerases β and λ and Increases Bleomycin Sensitivity of Human Cancer Cells.

A major concept to sensitize cancer cells to DNA damaging agents is by inhibiting proteins in the DNA repair pathways. X-family DNA polymerases play critical roles in both base excision repair (BER) and nonhomologous end joining (NHEJ). In this study, we examined the effectiveness of honokiol to inhibit human DNA polymerase β (pol β), which is involved in BER, and DNA polymerase λ (pol λ), which is involved in NHEJ. Kinetic analysis with purified polymerases showed that honokiol inhibited DNA polymerase activity. The inhibition mode for the polymerases was a mixed-function noncompetitive inhibition with respect to the substrate, dCTP. The X-family polymerases, pol β and pol λ, were slightly more sensitive to inhibition by honokiol based on the Ki value of 4.0 μM for pol β, and 8.3 μM for pol λ, while the Ki values for pol η and Kf were 20 and 26 μM, respectively. Next we extended our studies to determine the effect of honokiol on the cytotoxicity of bleomycin and temozolomide in human cancer cell lines A549, MCF7, PANC-1, UACC903, and normal blood lymphocytes (GM12878). Bleomycin causes both single strand DNA damage that is repaired by BER and double strand breaks that are repaired by NHEJ, while temozolomide causes methylation damage repaired by BER and O6-alkylguanine-DNA alkyltransferase. The greatest effects were found with the honokiol and bleomycin combination in MCF7, PANC-1, and UACC903 cells, in which the EC50 values were decreased 10-fold. The temozolomide and honokiol combination was less effective; the EC50 values decreased three-fold due to the combination. It is hypothesized that the greater effect of honokiol on bleomycin is due to inhibition of the repair of the single strand and double strand damage. The synergistic activity shown by the combination of bleomycin and honokiol suggests that they can be used as combination therapy for treatment of cancer, which will decrease the therapeutic dosage and side effects of bleomycin.

Polyprenylated Benzoylphloroglucinols with DNA Polymerase Inhibitory Activity from the Fruits of Garcinia schomburgkiana.

Chemical investigation of the fruits of Garcinia schomburgkiana collected in Vietnam led to the isolation of eight new schomburgkianones, A-H (1-8), four known (9-12) polyprenylated benzoylphloroglucinols, and four known biflavonoids. The structures of these compounds were elucidated by spectroscopic and chemical means. The absolute configuration at C-40 of 1 and 2 was determined by (1)H NMR analyses of their MPA esters. The configuration of the bicyclo[3.3.1]nonane core of the polyprenylated benzoylphloroglucinols was assigned by comparison of their experimental ECD spectra with those of related compounds. The polyprenylated benzoylphloroglucinols exhibited inhibitory activities against mammalian DNA polymerases α and λ, with IC50 values ranging from 5.0 to 8.8 μM. Compounds 1, 2, 4, 5, and 9-11 showed cytotoxic effects against HeLa human cervical cancer cells with median lethal dose values lower than 10 μM.

A repeat protein-based DNA polymerase inhibitor for an efficient and accurate gene amplification by PCR.

A polymerase chain reaction (PCR) using a thermostable DNA polymerase is the most widely applied method in many areas of research, including life sciences, biotechnology, and medical sciences. However, a conventional PCR incurs an amplification of undesired genes mainly owing to non-specifically annealed primers and the formation of a primer-dimer complex. Herein, we present the development of a Taq DNA polymerase-specific repebody, which is a small-sized protein binder composed of leucine rich repeat (LRR) modules, as a thermolabile inhibitor for a precise and accurate gene amplification by PCR. We selected a repebody that specifically binds to the DNA polymerase through a phage display, and increased its affinity to up to 10 nM through a modular evolution approach. The repebody was shown to effectively inhibit DNA polymerase activity at low temperature and undergo thermal denaturation at high temperature, leading to a rapid and full recovery of the polymerase activity, during the initial denaturation step of the PCR. The performance and utility of the repebody was demonstrated through an accurate and efficient amplification of a target gene without nonspecific gene products in both conventional and real-time PCRs. The repebody is expected to be effectively utilized as a thermolabile inhibitor in a PCR. Biotechnol. Bioeng. 2016;113: 2544-2552. © 2016 Wiley Periodicals, Inc.

Label-free molecular beacon for real-time monitoring of DNA polymerase activity.

Traditional methods for assaying DNA polymerase activity are discontinuous, time consuming, and laborious. Here, we report a new approach for label-free and real-time monitoring of DNA polymerase activity using a Thioflavin T (ThT) probe. In the presence of DNA polymerase, the DNA primer could be elongated through polymerase reaction to open MB1, leading to the release of the G-quartets. These then bind to ThT to form ThT/G-quadruplexes with an obvious fluorescence generation. It exhibits a satisfying detection result for the activity of DNA polymerase with a low detection limit of 0.05 unit/ml. In addition, no labeling with a fluorophore or a fluorophore-quencher pair is required; this method is fairly simple, fast, and low cost. Furthermore, the proposed method was also applied to assay the inhibition of DNA polymerase activity. This approach may offer potential applications in drug screening, clinical diagnostics, and some other related biomedical research.

5-O-Acyl plumbagins inhibit DNA polymerase activity and suppress the inflammatory response

We previously found that vitamin K3 (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of human mitochondrial DNA polymerase (pol) γ. In this study, we focused on plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), and chemically synthesized novel plumbagins conjugated with C2:0 to C22:6 fatty acids (5-O-acyl plumbagins). These chemically modified plumbagins displayed enhanced mammalian pol inhibition, with plumbagin conjugated to docosahexaenoic acid (C22:6-acyl plumbagin) exhibiting the strongest inhibition of pol λ among the ten 5-O-acyl plumbagins synthesized. C22:6-acyl plumbagin selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols or DNA metabolic enzymes tested. The inhibition of pol λ, a DNA repair/recombination pol, by these compounds was significantly correlated with both their suppression of lipopolysaccharide (LPS) induced tumor necrosis factor-α (TNF-α) production by mouse RAW264.7 macrophages and the reduction of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in the mouse ear. These data indicate that 5-O-acyl plumbagins act as anti-inflammatory agents by inhibiting mammalian pol λ. These results further suggest that C22:6-acyl plumbagin is a promising anti-inflammatory candidate and that acylation could be an effective chemical modification to improve the anti-inflammatory activity of vitamin K3 derivatives, such as plumbagin.

DNA Synthesis Is Required for Reprogramming Mediated by Stem Cell Fusion

SummaryEmbryonic stem cells (ESCs) can instruct the conversion of differentiated cells toward pluripotency following cell-to-cell fusion by a mechanism that is rapid but poorly understood. Here, we used centrifugal elutriation to enrich for mouse ESCs at sequential stages of the cell cycle and showed that ESCs in S/G2 phases have an enhanced capacity to dominantly reprogram lymphocytes and fibroblasts in heterokaryon and hybrid assays. Reprogramming success was associated with an ability to induce precocious nucleotide incorporation within the somatic partner nuclei in heterokaryons. BrdU pulse-labeling experiments revealed that virtually all successfully reprogrammed somatic nuclei, identified on the basis of Oct4 re-expression, had undergone DNA synthesis within 24 hr of fusion with ESCs. This was essential for successful reprogramming because drugs that inhibited DNA polymerase activity effectively blocked pluripotent conversion. These data indicate that nucleotide incorporation is an early and critical event in the epigenetic reprogramming of somatic cells in experimental ESC-heterokaryons.

Isolation and Characterization of High Affinity Aptamers Against DNA Polymerase Iota

Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.

Synthetic nucleotides as probes of DNA polymerase specificity.

The genetic code is continuously expanding with new nucleobases designed to suit specific research needs. These synthetic nucleotides are used to study DNA polymerase dynamics and specificity and may even inhibit DNA polymerase activity. The availability of an increasing chemical diversity of nucleotides allows questions of utilization by different DNA polymerases to be addressed. Much of the work in this area deals with the A family DNA polymerases, for example, Escherichia coli DNA polymerase I, which are DNA polymerases involved in replication and whose fidelity is relatively high, but more recent work includes other families of polymerases, including the Y family, whose members are known to be error prone. This paper focuses on the ability of DNA polymerases to utilize nonnatural nucleotides in DNA templates or as the incoming nucleoside triphosphates. Beyond the utility of nonnatural nucleotides as probes of DNA polymerase specificity, such entities can also provide insight into the functions of DNA polymerases when encountering DNA that is damaged by natural agents. Thus, synthetic nucleotides provide insight into how polymerases deal with nonnatural nucleotides as well as into the mutagenic potential of nonnatural nucleotides.

Review of QSAR for DNA Polymerase Inhibitors and New Models for Heterogeneous Series of Compounds

DNA polymerases are essential enzymes for DNA replication, repair and recombination. The high number of possible candidates creates the necessity of Quantitative Structure-Activity Relationship models in order to guide the DNA polymerase inhibitors. In this work, we revised different computational studies for a very large and heterogeneous series of DNA polymerase inhibitors. First, we reviewed QSAR methods with different compounds to find out the structural requirements for DNA polymerase inhibitory activity. Last, we report a new LDA analysis with the different molecular descriptors calculated with DRAGON software.

Pyrovanadolysis, a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate, Mn2+, and DNA Polymerase of Bacteriophage T7

DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP(i)). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP(i), a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP(i) complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn(2+), larger than Mg(2+), fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.

An optimized sensitive method for quantitation of DNA/RNA viruses in heparinized and cyropreserved plasma

Sodium heparin, an anticoagulant used widely for blood collection, has been known to inhibit DNA polymerase activity in polymerase chain reaction (PCR) assays. However, all cryopreserved plasma samples collected in the 1980s and early 1990s at the Multicenter AIDS Cohort Study were from heparin-treated blood, which poses a problem in quantifying the target nucleic acids contained in those samples by PCR assay. In this study, a nucleic acid extraction procedure was optimized to remove the heparin from extracted nucleic acids. Using this optimized method, similar human immunodeficiency virus 1 (HIV-1) and cytomegalovirus (CMV) loads of these viruses that were added to normal donor blood from ethylenediaminetetraacetic acid (EDTA), acid citrate dextrose (ACD) or sodium heparin tubes were detected by reverse transcriptase (RT) real-time PCR and real-time PCR. Comparable HIV-1 and CMV loads were also detected in the blood of persons with active HIV-1 and CMV infections collected in EDTA-, ACD- or sodium heparin-treated tubes by RT real-time and real-time PCR. The findings showed that the optimized nucleic acid extraction procedure efficiently removes the heparin inhibition effect on the performance of real-time PCR. This method could be used to extract nucleic acids from archived, heparinized plasma for PCR based quantitation of target molecules.

UBE2M-mediated p27 Kip1 degradation in gemcitabine cytotoxicity

Gemcitabine (2′-deoxy-2′, 2′-difluorocytidine; Gem) is a nucleoside anti-metabolite and is commonly used for treating various human cancers including human bladder carcinoma. Gemcitabine not only functions as a suicide nucleoside analog but also inhibits DNA polymerase activity and results in the termination of chain elongation. Using 2-dimensional gel electrophoresis analysis, a Gem-induced protein was identified as UBE2M (a.k.a. UBC12), a NEDD8 conjugation E2 enzyme which contributes to protein degradation. Gem induced UBE2M expression at both RNA and protein levels in several human cancer cell lines. The induction of UBE2M by Gem was accompanied by a reduction in p27 Kip1 protein levels, which could be restored by silencing UBE2M expression with siRNA or by treating cells with the proteasome inhibitor MG132, indicating that UBE2M mediates Gem-induced p27 Kip1 protein degradation. The induction of UBE2M and reduction of p27 Kip1 by Gem were prevented by the PI3K inhibitor LY294002. These results indicate that PI3K activity is necessary for Gem-induced UBE2M expression and that UBE2M facilitates degradation of p27 Kip1. Notably, silencing of UBE2M expression reduced Gem sensitivity in NTUB1 cells, suggesting that UBE2M mediates in part cell sensitivity to Gem, possibly by degradation of p27 Kip1. Analysis of Gem-resistant sub lines also showed that loss of UBE2M and increased p27 Kip1 expression were associated with the acquisition of drug resistance. In conclusion, our results demonstrate a role for UBE2M in mediating cytotoxicity of gemcitabine in human urothelial carcinoma cells while also suggesting a potential function of p27 Kip1 in drug resistance.

Anti-cancer Effect of Spinach Glycoglycerolipids as Angiogenesis Inhibitors Based on the Selective Inhibition of DNA Polymerase Activity

Plants contain major glycoglycerolipids, such as monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG), in the chloroplast membrane. The bioactivities of purified MGDG, DGDG and SQDG from spinach have been investigated extensively. MGDG and SQDG have been shown to inhibit the activities of mammalian DNA polymerases, but DGDG has no such inhibitory effect. The effect of these glycoglycerolipids on cancer cells, angiogenesis and solid tumor growth might be mediated via their inhibition of replicative DNA polymerase activities. On the basis of these findings, we discuss the mode of action of plant chloroplast glycoglycerolipids as anti-cancer therapeutic agents.