Establishment of a system for finding inhibitors of ε RNA binding with the HBV polymerase.

Although several nucleo(s)tide analogs are available for treatment of HBV infection, long-term treatment with these drugs can lead to the emergence of drug-resistant viruses. Recent HIV-1 studies suggest that combination therapies using nucleo(s)tide reverse transcriptase inhibitors (NRTIs) and non-nucleo(s)tide reverse transcriptase inhibitors (NNRTIs) could drastically inhibit the viral genome replication of NRTI-resistant viruses. In order to carry out such combinational therapy against HBV, several new NRTIs and NNRTIs should be developed. Here, we aimed to identify novel NNRTIs targeting the HBV polymerase terminal protein (TP)-reverse transcriptase (RT) (TP-RT) domain, which is a critical domain for HBV replication. We expressed and purified the HBV TP-RT with high purity using an Escherichia coli expression system and established an in vitro ε RNA-binding assay system. Then, we used TP-RT in cell-free assays to screen candidate inhibitors from a chemical compound library, and identified two compounds, 6-hydroxy-DL-DOPA and N-oleoyldopamine, which inhibited the binding of ε RNA with the HBV polymerase. Furthermore, these drugs reduced HBV DNA levels in cell-based assays as well by inhibiting packaging of pregenome RNA into capsids. The novel screening system developed herein should open a new pathway the discovery of drugs targeting the HBV TP-RT domain to treat HBV infection.

tBHQ-induced HO-1 expression is mediated by calcium through regulation of Nrf2 binding to enhancer and polymerase II to promoter region of HO-1.

Induction of Nrf2-mediated detoxifying/antioxidant enzymes is an effective strategy for cancer chemoprevention. The goal of this study was to examine the role of calcium [Ca(2+)] in regulating a well-known phenolic chemopreventive compound tertiary-butylhydroquinone (tBHQ) activation of Nrf2 and induction of Nrf2 downstream target gene heme-oxygenase (HO-1). tBHQ alone caused Nrf2 nuclear localization and induced HO-1 mRNA and protein expression in a dose-dependent manner. Using RT-PCR and Western blotting, we showed that tBHQ-induced transcription of HO-1 is Ca(2+)-dependent. Chelation of [Ca(2+)](ext) or [Ca(2+)](intra) by EGTA or BAPTA attenuated tBHQ-induced HO-1. Cotreatment of tBHQ with inhibitors of [Ca(2+)]-sensitive protein kinase C and camodulin kinase did not attenuate HO-1 induction. Nuclear translocation of Nrf2 induced by tBHQ was also not affected by treatment of EGTA or BAPTA. Additionally, EGTA and BAPTA treatments decreased basal nuclear phosphorylation of CREB and decreased tBHQ-induced Nrf2-CBP binding and Nrf2 binding to enhancer as well as polymerase II binding to the promoter of HO-1 gene. Furthermore, tBHQ in combination with higher [Ca(2+)](ext) augmented HO-1 induction both in vitro and in vivo, indicating that the modulation of [Ca(2+)](int) could be used as an adjuvant to increase the efficacy of chemopreventive agents. Taken together, our results indicated that in addition to tBHQ-induced oxidative stress-mediated Nrf2 translocation, HO-1 induction by tBHQ also appears to be dependent on a series of Ca(2+)-regulated mechanisms.

Comparison of hyperthermia radiosensitization and DNA polymerase inactivation in human normal and melanoma cell lines of different radiosensitivities.

Two human melanoma cell lines (one radiosensitive, HT144 and one radioresistant, SK Mel-3) and one normal human fibroblast (AG1522) were evaluated for thermal radiosensitization and the thermal enhancement ratios (TERs) were calculated. These were compared with residual polymerase activity to determine if this activity could be used to predict TERs. In all three cell lines, there was a good correlation between TER and residual polymerase alpha or beta activity. Polymerase beta was more sensitive than polymerase alpha as an indicator for TER. There were small cell line-dependent differences (not related to radiosensitivity) among the correlation curves, indicating that for each cell/tumor-type polymerase activity, vs. TER may have to be calibrated.

Effects of gypenosides on mouse splenic lymphocyte transformation and DNA polymerase II activity in vitro.

AIM: To study the effects of gypenosides (Gyp) on lymphocyte transformation and DNA polymerase II activity. METHODS: Lymphocyte transformation response was induced by concanavalin A and lipopolysaccharides respectively. The activity of DNA polymerase II and DNA synthesis were assayed with TTP and [3H]TdR incorporation respectively in mixed lymphocyte culture test. RESULTS: Gyp 2.5-20 mg L-1 enhanced splenic T- and B- cell transformation, increased the DNA synthesis and potentiated the activity of DNA polymerase II. However, Gyp > 40 mg L-1 showed contrary effects. CONCLUSION: Gyp regulated lymphocyte transformation and DNA synthesis by regulating DNA polymerase II activity.

[Effects of Ganoderma polysaccharides on the activity of DNA polymerase alpha of splenocytes and immune function in aged mice].

The activity of DNA polymerase alpha in splenocytes of 24-month-old mice was about 35.6% lower than that of 3-month-old mice. Aged mice were intraperitoneally administered Ganoderma polysaccharides (GL-B) once a day for 4 days and then the activity of the enzyme was assessed. The results showed that GL-B at doses of 25 and 50 mg/kg-1 enhanced the activity of the enzyme in aged mouse splenocytes by 44.0 and 58.8% respectively. In addition, the mixed lymphocyte response to alloantigen, automatic proliferation and IL-2 production of splenocytes in aged mice declined as compared with that in young adult mice. GL-B (50, 100, 200 micrograms.ml-1) was found to restore those parameters to the levels of that of young mice in vitro.

Incorporation of cytosine arabinoside monophosphate into DNA at internucleotide linkages by human DNA polymerase alpha.

The incorporation of cytosine arabinoside monophosphate (araCMP) into DNA at internucleotide linkages by DNA polymerase alpha (DNA pol alpha) has been investigated by using oligonucleotide primed DNA templates. The products of reactions catalyzed by DNA pol alpha in vitro were analyzed on polyacrylamide gels to measure insertion of araCMP, extension from an araCMP 3' terminus, and binding of the enzyme to an araCMP 3' terminus. The results show that insertion of araCMP opposite dGMP in the DNA template is about 3-fold less efficient than insertion of dCMP. Extension from an araCMP 3' terminus by addition of the next complementary nucleotide is approximately 2000-fold less efficient than extension from a correctly base-paired 3' terminus. In the absence of the second substrate, dNTP, DNA pol alpha binds with approximately equal affinities to DNA templates that contain oligonucleotide primers with araCMP or dCMP positioned at the 3' terminus. In the presence of dNTP, the enzyme extends the araCMP 3' terminus or dissociates, but it is not trapped at the araCMP 3' terminus in a nonproductive ternary complex as is observed at the ddCMP 3' terminus. To determine if slow phosphodiester bond formation contributes to the observed extension rate from the araCMP 3' terminus by DNA pol alpha, oligonucleotide primers with araCMP positioned at the 3' terminus were elongated by addition of the alpha-phosphorothioate analogue of the next complementary nucleotide. The rate of extension from araCMP by addition of 2'-deoxyadenosine 5'-O-phosphorothioate (dAMP alpha S) was 6-fold slower than by addition of dAMP, indicating that bond formation is partially rate limiting in the extension reaction. Thus, inefficient extension from the araCMP 3' terminus is the major determinant contributing to the low incorporation frequency of araCMP into DNA by DNA pol alpha, and this inefficiency can be attributed, in part, to slower phosphodiester bond formation at the araCMP 3' terminus.

[Modification of human DNA polymerase alpha by N-acetylimidazole]. / Modifikatsiia DNK-polimerazy alpha cheloveka N-atsetilimidazolom.

The modification of tyrosine residues of the human placenta DNA-polymerase alpha by N-acetylimidazole was investigated. The poly(dT)-template and the r(pA)10-primer a each added separately or simultaneously do not influence the rate of enzyme inactivation. In the presence of poly(dT)-r(pA)10 no effect of dCTP and dTTP (noncomplementary to template) and of dAMP and dADP (complementary to template) on the rate and the level of the enzyme inactivation was found. However dATP revealed practically complete protection. Orthophosphate, pyrophosphate each taken separately do not influence the rate of enzyme inactivation with this reagent. The presence of dADP with either ortho- or pyrophosphate, or dAMP with the one of these ligands leads to half protective action in comparison with dATP. Imidazolides of phosphonoacetic acid and 5'-adenylyl++ 1(phosphonoacetic acid) do not inactivate DNA-polymerase alpha from human placenta and the Klenov fragment of DNA-polymerase I from E. coli. All data obtained allow to suggest that the tyrosine residue in the dNTP binding site of DNA-polymerase reveals stacking with the nucleotide only if dNTP is complementary to the template.

Protein-nucleic acid interaction in reactions catalyzed with DNA polymerases.

The affinities of oligothymidylates and of some analogs for the template site, of a set of oligodeoxyribo- and oligoribonucleotides for the primer site, and of dNTPs and some analogs for the substrate sites of DNA polymerase I Klenow fragment and of human placenta DNA polymerase alpha were measured using them either as competitors of affinity modification or as substrates. The data obtained enable us to hypothesize that the Me2+-dependent electrostatic contact and hydrogen bond of a single internucleotide phosphate and the hydrophobic interactions of the other nucleotide units determine the formation of oligonucleotide-template site complexes. Interaction of the primer's 3'-terminal hydroxy group and of the negatively charged adjacent phosphate with the enzyme, and Watson-Crick base pairing with the template are of crucial importance for the formation of the ternary enzyme-template-primer complex. dNTP and dNMP imidazolides inactivate enzymes via an affinity modification mechanism only in the presence of the template-primer complex. dNTP affinities exceed those of dNDPs and dNMPs, the enhancement being most significant for the substrate that is complementary to the template, thus suggesting the participation of the gamma-phosphate of dNTP in the substrate selection step.

DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA.

Unique single-stranded regions of simian virus 40 DNA, phage M13 virion DNA, and several homopolymers were used as templates for the synthesis of (p)ppRNA-DNA chains by CV-1 cell DNA primase-DNA polymerase alpha. Intact RNA primers, specifically labeled with an RNA capping enzyme, were typically 6 to 8 ribonucleotides long, although their lengths ranged from 1 to 9 bases. The fraction of intact RNA primers 1 to 4 ribonucleotides long was 14 to 73%, depending on the template used. RNA primer length varied among primers initiated at the same nucleotide, as well as with primers initiated at different sites. Thus, the size of an RNA primer depended on template sequence. Initiation sites were identified by mapping 5' ends of nascent RNA-DNA chains on the template sequence, identifying the 5'-terminal ribonucleotide, and partially sequencing one RNA primer. A total of 56 initiation events were identified on simian virus 40 DNA, an average of 1 every 16 bases. Some sites were preferred over others. A consensus sequence for initiation sites consisted of either 3'-dCTTT or 3'-dCCC centered within 7 to 25 pyrimidine-rich residues; the 5' ends of RNA primers were complementary to the dT or dC. High ATP/GTP ratios promoted initiation of RNA primer synthesis at 3'-dCTTT sites, whereas low ATP/GTP ratios promoted initiation at 3'-dCCC sites. Similarly, polydeoxythymidylic acid and polydeoxycytidylic acid were the only effective homopolymer templates. Thus, both template sequence and ribonucleoside triphosphate concentrations determine which initiation sites are used by DNA primase-DNA polymerase alpha. Remarkably, initiation sites selected in vitro were strikingly different from initiation sites selected during simian virus 40 DNA replication in vivo.

On the fidelity of DNA replication. Studies with human placenta DNA polymerases.

The fidelity of DNA synthesis with purified DNA polymerase alpha and beta from human placenta has been studied. With poly[d(A-T)] as the template-primer and Mg2+ as the metal activator, DNA polymerase alpha incorporates 1 mol of dGMP for every 6,000 to 12,000 mol of complementary nucleotides polymerized. Under the same conditions, DNA polymerase beta is more accurate, the error rate being 1/20,000 to 1/60,000. This greater accuracy of DNA polymerase beta is observed with a variety of homopolymer templates. With both enzymes, substitution of Mg2+ with activating concentrations of Mn2+ or Co2+ enhances the frequency of misincorporation. At greater than activating concentrations of Mn2+ and Co2+, there is an inhibition of complementary nucleotide incorporation, further increasing the frequency of misincorporation. Nearest neighbor analysis of the products synthesized with both enzymes indicates that the noncomplementary nucleotides are incorporated predominantly as single base substitutions. The greater accuracy of DNA polymerase beta over DNA polymerase alpha should be considered in relationship to their possible roles in DNA replication and repair.

Inhibition of DNA polymerase from L1210 murine leukemia by a sulfhydryl reagent from agaricus bisporus.

The 490 quinone, a natural sulfhydryl-arylating reagent from the mushroom, Agaricus bisporus, markedly inhibited L1210 murine leukemia DNA polymerase alpha while resulting in little inhibition of DNA polymerase beta from this source. This quinone was more strongly inhibitory than p-chloromercuri-benzoate or N-ethylmaleimide and was less readily neutralized by sulfhydryl-containing molecules such as dithioerythritol. Preliminary experiments indicate that DNA protects DNA polymerase alpha from inhibition by the 490 quinone. The inhibition of DNA synthesis by quinone 490 may contribute significantly to the cytotoxicity of this compound and to the potential of gamma-L-glutaminyl-4-hydroxybenzene as an antitumor agent.