Pol Activity Research Articles

Silencing SIRT1 promotes the anti-HBV action of IFN-α by regulating Pol expression and activating the JAK-STAT signaling pathway

PurposeThe purpose this study is to investigate the impact of SIRT1 on the anti-HBV activity of IFN-α and further elucidate its underlying mechanism. MethodsHepG2.2.15 cells stably transfected with HBV virus were chosen as the primary study subject. IFN-α was used to stimulate the cells and regulate the expression of SIRT1, and the JAK-STAT pathway and HBV-related indices were measured by qRT-PCR, Western blotting and ELISA. Immunofluorescence (IF) was used to detect the nuclear translocation of STAT1 and STAT2. Coimmunoprecipitation (Co-IP) was used to detect the binding of SIRT1 to HBV Polymerase (Pol). ResultsIn HepG2.2.15 cells, we found changes in SIRT1 expression. We show that silencing SIRT1 promotes the IFN-α-triggered Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway and consequently enhances the antiviral effects of IFN-α against HBV replication. Importantly, SIRT1 can interact with Pol and increase JAK-STAT activity by regulating Pol expression. Additionally, the inhibition of SIRT1 activity by treatment with the SIRT1 inhibitor selisistat enhanced the anti-HBV effect of IFN-α and JAK-STAT pathway activity. ConclusionIn conclusion, our results demonstrate that silencing SIRT1 activates the JAK-STAT pathway and enhances the anti-HBV activity of IFN-α by inhibiting Pol expression. This would be a promising therapeutic target to improve the efficacy of IFN-α in the treatment of CHB.

Reactivation of Epstein-Barr Virus from Latency Involves Increased RNA Polymerase Activity at CTCF Binding Sites on the Viral Genome.

The ability of Epstein-Barr virus (EBV) to switch between latent and lytic infection is key to its long-term persistence, yet the molecular mechanisms behind this switch remain unclear. To investigate transcriptional events during the latent-to-lytic switch, we utilized Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to map cellular RNA polymerase (Pol) activity to single-nucleotide resolution on the host and EBV genome in three different models of EBV latency and reactivation. In latently infected Mutu-I Burkitt lymphoma (BL) cells, Pol activity was enriched at the Qp promoter, the EBER region, and the BHLF1/LF3 transcripts. Upon reactivation with phorbol ester and sodium butyrate, early-phase Pol activity occurred bidirectionally at CTCF sites within the LMP-2A, EBER-1, and RPMS1 loci. PRO-Seq analysis of Akata cells reactivated from latency with anti-IgG and a lymphoblastoid cell line (LCL) reactivated with small molecule C60 showed a similar pattern of early bidirectional transcription initiating around CTCF binding sites, although the specific CTCF sites and viral genes were different for each latency model. The functional importance of CTCF binding, transcription, and reactivation was confirmed using an EBV mutant lacking the LMP-2A CTCF binding site. This virus was unable to reactivate and had disrupted Pol activity at multiple CTCF binding sites relative to the wild-type (WT) virus. Overall, these data suggest that CTCF regulates the viral early transcripts during reactivation from latency. These activities likely help maintain the accessibility of the viral genome to initiate productive replication. IMPORTANCE The ability of EBV to switch between latent and lytic infection is key to its long-term persistence in memory B cells, and its ability to persist in proliferating cells is strongly linked to oncogenesis. During latency, most viral genes are epigenetically silenced, and the virus must overcome this repression to reactivate lytic replication. Reactivation occurs once the immediate early (IE) EBV lytic genes are expressed. However, the molecular mechanisms behind the switch from the latent transcriptional program to begin transcription of the IE genes remain unknown. In this study, we mapped RNA Pol positioning and activity during latency and reactivation. Unexpectedly, Pol activity accumulated at distinct regions characteristic of transcription initiation on the EBV genome previously shown to be associated with CTCF. We propose that CTCF binding at these regions retains Pol to maintain a stable latent chromosome conformation and a rapid response to various reactivation signals.

Eukaryotic RNA Polymerase ⍺‐like Subunits Heterodimerize by Distinct Interaction Mechanisms

DNA‐dependent RNA Polymerases (Pols) are present in every living cell and even encoded by some viruses. Pols are responsible for the process of transcription, and Pols from the three domains of life are constructed of a conserved core complex that includes a dimer of α or α‐like subunits that serve as a scaffold for Pol assembly. Bacteria and archaea each encode a single Pol that transcribes all forms of RNA, while eukaryotes encode three specialized Pols (I‐III) containing one of two distinct α‐like heterodimers. One α‐like heterodimer is shared between Pols I and III, while there is a paralogous Pol II heterodimer. The α‐like subunits are clinically relevant as mutations in the Pol I/III heterodimer are associated with Treacher Collins Syndrome and 4H Leukodystrophy, while mutations in the Pol II heterodimer are associated with Primary Ovarian Insufficiency. These mutations often result in defects in Pol assembly and/or activity, although the vast majority remain uncharacterized. It is currently unclear if heterodimer formation is functionally similar in α‐like subunit orthologs and/or paralogs and how these similarities and differences can be used to study the α‐like subunit associated diseases. To examine this, we mutated several regions of the yeast and human small α‐like subunits to test their contribution to heterodimer interaction using several types of biochemical and genetic assays. Here we show that different regions serve differential roles in heterodimerization, in a polymerase‐ and species‐specific manner, with both human subunits being more sensitive to mutations. This suggests that although the subunits are evolutionary conserved, they interact differently. More broadly, these findings help explain why some disease mutations have little to no effect in yeast and possibly other model systems, and may inform us how to make better disease models in the future.

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.

Chimeric Phi29 DNA polymerase with helix-hairpin-helix motifs shows enhanced salt tolerance and replication performance.

SummaryPhi29 DNA polymerase (Phi29 Pol) has been successfully applied in DNA nanoball‐based sequencing, real‐time DNA sequencing from single polymerase molecules and nanopore sequencing employing the sequencing by synthesis (SBS) method. Among these, polymerase‐assisted nanopore sequencing technology analyses nucleotide sequences as a function of changes in electrical current. This ionic, current‐based sequencing technology requires polymerases to perform replication at high salt concentrations, for example 0.3 M KCl. Nonetheless, the salt tolerance of wild‐type Phi29 Pol is relatively low. Here, we fused helix–hairpin–helix (HhH)2 domains E‐L (eight repeats in total) of topoisomerase V (Topo V) from the hyperthermophile Methanopyrus kandleri to the Phi29 Pol COOH terminus, designated Phi29EL DNA polymerase (Phi29EL Pol). Domain fusion increased the overall enzyme replication efficiency by fourfold. Phi29EL Pol catalysed rolling circle replication in a broader range of salt concentrations than did Phi29 Pol, extending the KCl concentration range for activity up to 0.3 M. In addition, the mutation of Glu375 to Ser or Gln increased Phi29EL Pol activity in the presence of KCl. In this work, we produced a salt‐tolerant Phi29 Pol derivative by means of (HhH)2 domain insertion. The multiple advantages of this insertion make it a good substitute for Phi29 Pol, especially for use in nanopore sequencing or other circumstances that require high salt concentrations.

The study of the antioxidant activity of polyphenol extracts from cranberry leaves on the model of insulin resistance

Topicality. In Ukraine, the number of patients with diabetes is growing rapidly, it ranks the 4th in the structure of diseases of the endocrine system, and it is one of the main causes of incapacitation and disability among the population and takes the second place by the number of deaths. Polyphenolic extracts from leaves of the heather (Ericaceae) family, in particular blueberries, cranberries and bearberry, in the previous studies demonstrated the presence of antioxidant, hypoglycemic and lipotropic properties. However, the pharmacological activity of extracts from cranberry leaves has not been sufficiently studied. Aim. To study the effect of polyphenolic extracts from cranberry leaves on the processes of LPO and the state of the antioxidant defense in the liver and serum in rats with the experimental insulin resistance. Materials and methods. The study was performed on white outbred male rats weighing 180 ± 15 g, kept in the vivarium of the NUPh. Insulin resistance was induced by keeping animals on a high fructose diet for 6 weeks according to the recommendations. The extracts were administered intragastrically in the dose of 200 μg/100 g of the body weight from the fourth week of the experiment for 14 days. The POL activity was assessed by the level of TBA reactants, diene conjugates and conjugated trienes in the liver and serum. The state of the antioxidant defense was assessed by the level of reduced glutathione, as well as the activity of catalase and superoxide dismutase. Indicators were determined by the spectrophotometric method. Results and discussion. The development of the experimental resistance was accompanied by an increase in LPS activity in the liver and serum of rats. In the serum of animals with IR the content of primary products of LPO increased in 1.75 times, the content of triene conjugates in 1.7 times, respectively, while the content of TBA reactants in 1.61 times. The introduction of the polyphenolic concentrate from cranberry leaves was accompanied by a sharp decrease in the products of lipid peroxidation both in the eserum and liver to almost ascending levels. There was normalization of the activity of enzymes of the first line of the antioxidant defense compared to animals with insulin resistance due to the presence of plant flavonoids in the extract. Conclusions. Thus, the data obtained indicate that the polyphenolic extract from cranberry leaves exhibits antioxidant properties in the liver and serum of rats with the experimental insulin resistance. The polyphenolic extract also restored the antioxidant defense of cells and the body as a whole. The data obtained indicate the need for further research to create a new herbal medicine for use in the treatment of insulin resistance and diabetes.

Networked Communication between Polymerase and Exonuclease Active Sites in Human Mitochondrial DNA Polymerase.

High fidelity human mitochondrial DNA polymerase (Pol γ) contains two active sites, a DNA polymerization site (pol) and a 3'-5' exonuclease site (exo) for proofreading. Although separated by 35 Å, coordination between the pol and exo sites is crucial to high fidelity replication. The biophysical mechanisms for this coordination are not completely understood. To understand the communication between the two active sites, we used a statistical-mechanical model of the protein ensemble to calculate the energetic landscape and local stability. We compared a series of structures of Pol γ, complexed with primer/template DNA, and either a nucleotide substrate or a series of nucleotide analogues, which are differentially incorporated and excised by pol and exo activity. Despite the nucleotide or its analogues being bound in the pol, Pol γ residue stability varied across the protein, particularly in the exo domain. This suggests that substrate presence in the pol can be "sensed" in the exo domain. Consistent with this hypothesis, in silico mutations made in one active site mutually perturbed the energetics of the other. To identify specific regions of the polymerase that contributed to this communication, we constructed an allosteric network connectivity map that further demonstrates specific pol-exo cooperativity. Thus, a cooperative network underlies energetic connectivity. We propose that Pol γ and other dual-function polymerases exploit an energetic coupling network that facilitates domain-domain communication to enhance discrimination between correct and incorrect nucleotides.

Bayesian Nonparametric Analysis of Transcriptional Processes

Optical tweezers are routinely used to extract dynamics of nucleic acid associated motors, such as RNA polymerase (RNAP). Their ability to provide data with base-pair spatial resolution is the key. It gives us access to information encoded on the sequence-dependent mechanism underlying molecular motor processivity. Of particular interest is the statistics of RNA Polymerase's pause and dwell dependencies on DNA sequence during both the initiation and elongation stages of transcription. Inspired by results suggesting RNA Pol's DNA composition-specific transcriptional mechanism, we combine the highly temporally resolved data from optical traps with the novel statistical analysis methods of Bayesian nonparametrics (BNPs) to draw base-pair level insight on RNA Pol's activity. On account of BNPs' flexibility in recruiting models of RNA Pol dynamics warranted by the data, and RNA Pol's intrinsically complex dynamics, we rigorously constrain the models allowed by BNPs by proposing new modeling strategies inspired by BNP tools scarcely half a decade old.

The Establishment of an Assay to Measure DNA Polymerase-Catalyzed Repair of UVB-Induced DNA Damage in Skin Cells and Screening of DNA Polymerase Enhancers from Medicinal Plants

An in vitro assay method was established to measure the activity of cellular DNA polymerases (Pols) in cultured normal human epidermal keratinocytes (NHEKs) by modifying Pol inhibitor activity. Ultraviolet (UV) irradiation enhanced the activity of Pols, especially DNA repair-related Pols, in the cell extracts of NHEKs. The optimal ultraviolet B (UVB) exposure dose and culture time to upregulate Pols activity was 100 mJ/cm2 and 4-h incubation, respectively. We screened eight extracts of medicinal plants for enhancement of UVB-exposed cellular Pols activity using NHEKs, and found that rose myrtle was the strongest Pols enhancer. A Pols’ enhancement compound was purified from an 80% ethanol extract of rose myrtle, and piceatannol was isolated by spectroscopic analysis. Induction of Pol activity involved synergy between UVB irradiation and rose myrtle extract and/or piceatannol. Both the extract and piceatannol reduced UVB-induced cyclobutane pyrimidine dimer production, and prevented UVB-induced cytotoxicity. These results indicate that rose myrtle extract and piceatannol, its component, are potential photo-protective candidates for UV-induced skin damage.

Activity of DNA polymerase λ in spikelets of rice and maize

Involvement of DNA polymerase (pol) enzymes in meiotic DNA repair has not been clearly understood in plants. DNA polymerase λ is involved in short patch DNA synthesis and base excision repair in both plants and animals. The presence and activity of the pol λ enzyme was studied in a protein isolated from spikelets during flower development stages of rice (Oryza sativa L.) and maize (Zea mays L.). Western blot analysis showed a 2- to 2.5-fold higher accumulation of pol λ in spikelets than in shoots. Assays of pol and in-gel activity showed the dideoxynucleotide triphosphate sensitive pol λ enzyme in spikelets of both the plants. An enhanced presence of the enzyme and its high activity suggests an active role of pol λ in meiotic recombination during microspore development.

The C-terminal Domain of the DNA Polymerase Catalytic Subunit Regulates the Primase and Polymerase Activities of the Human DNA Polymerase α-Primase Complex

The initiation of DNA synthesis during replication of the human genome is accomplished primarily by the DNA polymerase α-primase complex, which makes the RNA-DNA primers accessible to processive DNA pols. The structural information needed to understand the mechanism of regulation of this complex biochemical reaction is incomplete. The presence of two enzymes in one complex poses the question of how these two enzymes cooperate during priming of DNA synthesis. Yeast two-hybrid and direct pulldown assays revealed that the N-terminal domain of the large subunit of primase (p58N) directly interacts with the C-terminal domain of the catalytic subunit of polα (p180C). We found that a complex of the C-terminal domain of the catalytic subunit of polα with the second subunit (p180C-p70) stimulated primase activity, whereas the whole catalytically active heterodimer of polα (p180ΔN-p70) inhibited RNA synthesis by primase. Conversely, the polα catalytic domain without the C-terminal part (p180ΔN-core) possessed a much higher propensity to extend the RNA primer than the two-subunit polα (p180ΔN-p70), suggesting that p180C and/or p70 are involved in the negative regulation of DNA pol activity. We conclude that the interaction between p180C, p70, and p58 regulates the proper primase and polymerase function. The composition of the template DNA is another important factor determining the activity of the complex. We have found that polα activity strongly depends on the sequence of the template and that homopyrimidine runs create a strong barrier for DNA synthesis by polα.

Large-Scale Production and Structural and Biophysical Characterizations of the Human Hepatitis B Virus Polymerase

Hepatitis B virus (HBV) is a major human pathogen that causes serious liver disease and 600,000 deaths annually. Approved therapies for treating chronic HBV infections usually target the multifunctional viral polymerase (hPOL). Unfortunately, these therapies--broad-spectrum antivirals--are not general cures, have side effects, and cause viral resistance. While hPOL remains an attractive therapeutic target, it is notoriously difficult to express and purify in a soluble form at yields appropriate for structural studies. Thus, no empirical structural data exist for hPOL, and this impedes medicinal chemistry and rational lead discovery efforts targeting HBV. Here, we present an efficient strategy to overexpress recombinant hPOL domains in Escherichia coli, purifying them at high yield and solving their known aggregation tendencies. This allowed us to perform the first structural and biophysical characterizations of hPOL domains. Apo-hPOL domains adopt mainly α-helical structures with small amounts of β-sheet structures. Our recombinant material exhibited metal-dependent, reverse transcriptase activity in vitro, with metal binding modulating the hPOL structure. Calcomine orange 2RS, a small molecule that inhibits duck HBV POL activity, also inhibited the in vitro priming activity of recombinant hPOL. Our work paves the way for structural and biophysical characterizations of hPOL and should facilitate high-throughput lead discovery for HBV. The viral polymerase from human hepatitis B virus (hPOL) is a well-validated therapeutic target. However, recombinant hPOL has a well-deserved reputation for being extremely difficult to express in a soluble, active form in yields appropriate to the structural studies that usually play an important role in drug discovery programs. This has hindered the development of much-needed new antivirals for HBV. However, we have solved this problem and report here procedures for expressing recombinant hPOL domains in Escherichia coli and also methods for purifying them in soluble forms that have activity in vitro. We also present the first structural and biophysical characterizations of hPOL. Our work paves the way for new insights into hPOL structure and function, which should assist the discovery of novel antivirals for HBV.

Inhibitory effects of α-mangostin on mammalian DNA polymerase, topoisomerase, and human cancer cell proliferation

We found that the ethanol extract of mangosteen (Garcinia mangostana L.) fruit rind had a strong inhibitory effect on mammalian DNA polymerase (pol) activity and isolated α-mangostin as a potent pol inhibitor from the extract. In this study, the inhibitory activities against mammalian pols by α-mangostin and its related five compounds, 3-isomangostin, xanthone, 9,10-anthraquinone, 9-anthracenecarboxylic acid, and anthracene, were investigated. α-Mangostin was the most potent inhibitor of the mammalian pol species among the tested compounds, with IC₅₀ values of 14.8-25.6 μM. This compound also inhibited human DNA topoisomerases (topos) I and II activities with IC₅₀ values of 15.0 and 7.5 μM, respectively, but did not inhibit the activities of other DNA metabolic enzymes tested. α-Mangostin also did not directly bind to double-stranded DNA as determined by thermal transition analysis. α-Mangostin was found to suppress human colon HCT116 carcinoma cell proliferation with an LC₅₀ of 18.5 μM, inhibit the activity of cellular topos, halt cell cycle in the G2/M phase, and induce apoptosis. These results suggest that decreased proliferation by α-mangostin may be a result of the inhibition of cellular topos rather than pols, and α-mangostin might be an anticancer chemotherapeutic agent.

Novel Compounds from Marine Microorganisms that Inhibit Mammalian DNA Polymerase

We screened for selective inhibitors of mammalian DNA polymerase (Pol) species from natural products. We then identified compounds that are potential anticancer agents based on Pol inhibition. In this review, the focus is on symbiotic microorganisms that grow on wild marine plants and which could be used as a new screening source for anticancer compounds. The fungal strains were isolated from wild marine plants such as seaweeds and sea mosses collected in Japan. Nine novel compounds were isolated from the cultured broth extracts of isolated fungal strains. These compounds showed bioactivities such as selective inhibition of mammalian Pol activity among 10 mammalian Pol species and suppression of proliferation of human cancer cells while having no effect on the growth of normal human cells. The probability of discovering novel compounds from such sources is high. Therefore, microorganisms derived from wild marine plants appear to be a good screening source for bioactive compounds compared with other naturally occurring materials. Keywords: Marine microorganism, Seaweeds, Sea mosses, DNA polymerase (E.C.2.7.7.7), Enzyme inhibitor, Cytotoxicity, Anticancer drug

Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol) activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT) activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR). Wild-type 3173 Pol contains a proofreading 3′-5′ exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth) and two-enzyme (MMLV RT/Taq) RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics.

A Dominant Role for an Error‐Prone DNA Polymerase in Recombination Drives Evolution

Homologous recombination (HR) repairs double‐strand breaks in DNA by directing replication to copy sequence information from a homologous donor DNA. Importantly, this mode of repair is considered error‐free which implies that only high‐fidelity DNA polymerases (pols) perform recombination‐directed replication (RDR). Yet in E. coli, error‐prone Y‐family translesion pols function with RecA recombinase, are highly upregulated in response to stress, and promote mutations following DSBs, which suggests a possible role for these mutagenic pols in HR. Here, we investigate E. coli translesion pol activity in HR by reconstituting RDR in vitro. We find that translesion pol IV is proficient in RDR even without the β‐processivity clamp, whereas translesion pol V lacks this activity. Notably, low‐fidelity pol IV initiates RDR more efficiently than high‐fidelity replicative pol III and is preferentially recruited to D‐loop recombination intermediates under stress response conditions. Unexpectedly, we find that pol III is inhibited in RDR due to its proofreading activity which is stimulated at D‐loops. Finally, we show that pol IV promotes mutations during RDR. These data demonstrate a dominant role for error‐prone pol IV in HR and elucidate the molecular basis of adaptive mutagenesis which facilitates evolution under conditions of stress.

Studies on Human DNA Polymerase ϵ and GINS Complex and Their Role in DNA Replication

Studies on Human DNA Polymerase ϵ and GINS Complex and Their Role in DNA Replication

Effect of cadmium on growth and respiration of barley plants grown under two temperature regimes

We studied cadmium effect on the respiratory pathways ratio in the organs of barley (Hordeum distichum L., cv. Novichok) plants grown in water culture at two temperature regimes. Mineral nutrients were supplied daily in exponentially increasing amounts in order to provide for steady-state growth. CdSO4 (30, 60, or 100 μmol/l) was added to nutrient solution at a single time in the beginning of the exponential growth period (19 days after germination). In further 6 days, the relative growth rate and biomass accumulation declined stronger with the increase in the cadmium concentration in plants grown at 13/8°C (day/night) than at 21/17°C (day/night). Cadmium suppressed root respiration (down to 60% of control) stronger than leaf respiration, and the roots manifested a higher sensitivity to the inhibitor of alternative oxidase, salicylhydroxamic acid. The respiratory pathways ratio in the roots occurred against the background of activated lipid peroxidation (POL). The highest POL activity and the highest proportion of alternative respiration pathway (AP) (up to 46% of total respiration) were observed in the roots in the presence of the highest cadmium concentration (100 μM) under lower temperature (13/8°C). Thus, high cadmium concentrations affected strongly the total rate of respiration and respiratory pathways ratio. Growth temperature modulated Cd effects on respiration. AP activation is one of the mechanisms for maintenance of root cell homeostasis under cadmium-induced stress.

Trans-Complementation of HBV rtM204I mutant replication by HBV wild-type polymerase

The function of the hepatitis B virus (HBV) wild-type (WT) polymerase (pol) expressed alone or in the context of the intact genome when interacting with HBV rtM204I in HepG2 cells was compared. We show that WT pol expression from a packaging-defective RNA can complement defective rtM204I pol activity resulting in increased levels of HBV replicative intermediates (RI). Analysis of the genetically marked genomes showed that this restoration resulted from trans-complementation, rather than recombination. In contrast, we demonstrate that enhanced levels of total HBV RI observed when cells were cotransduced with both WT and rtM204I baculoviruses were predominantly WT RI. In this case, WT pol was produced from a full-length pregenomic RNA (pgRNA). We conclude that the WT pol has the capacity to trans-complement the replication defect of rtM204I; however, when expressed from an authentic pgRNA, as in a mixed infection, pol may not trans-complement efficiently.

Cell cycle arrest triggered by conjugated eicosapentaenoic acid occurs through several mechanisms including G1 checkpoint activation by induced RPA and ATR expression

Background Conjugated eicosapentaenoic acid (cEPA) containing conjugated double bonds, which is prepared by alkaline treatment of eicosapentaenoic acid (EPA), selectively inhibited the activities of both mammalian DNA polymerases (pols) and human DNA topoisomerases (topos). Methods Human colon carcinoma cell line, HCT116, was cultured and performed drug and small interfering RNA (siRNA) treatment, flow cytometry analysis, BrdU incorporation analysis, and western blot analysis. Results The levels of bromodeoxyuridine (BrdU) incorporation labeling during DNA synthesis were decreased in time- and dose-dependent manners in HCT116 cells, treated with cEPA. The level of chromatin association of RPA70, a subunit of the single-stranded DNA (ssDNA)-binding protein, was increased following cEPA exposure, suggesting that the replication forks were stalled in response to inhibition of replicative pol activity by cEPA in the cells. cEPA also induced the activation of ataxia-telangiectasia and Rad3-related (ATR) protein in HCT116 cells, and activated the G1 checkpoint pathway in the cells, which was down-regulated by a small interfering RNA (siRNA) against ATR protein. Moreover, caffeine, a known ATR kinase inhibitor, abrogated the cEPA-induced G1 checkpoint in HCT116 cells. General significance cEPA could inhibit the activity of replicative pols, such as pols α, δ and ɛ, affect the DNA replication fork including ssDNA, and then activate the G1 checkpoint pathway by the induction of RPA and ATR expression levels in cancer cells.