Interactions of azidothymidine triphosphate with the cellular DNA polymerases alpha, delta, and epsilon and with DNA primase.

The interactions of azidothymidine triphosphate, the metabolically active form of the anti-AIDS drug azidothymidine (zidovudine), with the cellular DNA polymerases alpha, delta, and epsilon, as well as with the RNA primer-forming enzyme DNA primase were studied in vitro. DNA polymerase alpha was shown to incorporate azidothymidine monophosphate into a growing polynucleotide chain. This occurred 2000-fold slower than the incorporation of natural dTTP. Despite the ability of polymerase alpha to use azidothymidine triphosphate as an alternate substrate, this compound was only marginally inhibitory to the enzyme (Ki greater than 1 mM). Furthermore, the DNA primase activity associated with DNA polymerase alpha was barely inhibited by azidothymidine triphosphate (Ki greater than 1 mM). Inhibition was more pronounced for DNA polymerases delta and epsilon. The type of inhibition was competitive with respect to dTTP, with Ki values of 250 and 320 microM, respectively. No incorporation of azidothymidine monophosphate was detectable with these two DNA polymerases because their associated 3'- to 5'-exonuclease activities degraded primer molecules prior to any measurable elongation. Template-primer systems with a preformed 3'-azidothymidine-containing primer terminus inhibited the three replicative polymerases rather potently. DNA polymerase alpha was inhibited with a Ki of 150 nM and polymerases delta and epsilon with Ki values of 25 and 20 nM, respectively. The type of inhibition was competitive with respect to the unmodified substrate poly(dA).oligo(dT) for all DNA polymerases tested. Performed 3'-azidothymidine-containing primers hybridized to poly(dA) were rather resistant to degradation by the 3'- to 5'-exonuclease of DNA polymerases epsilon and more susceptible to the analogous activity that copurified with DNA polymerase delta. It is proposed that the repair of 3'-azidothymidine-containing primers might become rate-limiting for the process of DNA replication in cells that have been treated with azidothymidine triphosphate.     

Evans Blue and other dyes as protein tyrosine phosphatase inhibitors

Commonly used dyes including Evans Blue and Trypan Blue were examined for their inhibitory activities against protein tyrosine phosphatases (PTPases), all of them showed inhibition of PTPases with different potencies. Of the 13 dyes tested, four exhibited IC(50) value of less than 10 microM, Evans Blue lowest IC(50) of 1.3 microM against PTP1B. Care must be taken in the use of dyes for clinical or biochemical experiments to avoid unwanted side effects. Some of the low molecular weight dyes might be useful as lead compounds for the development of potent and selective PTPase inhibitors.     

An inhibitor of DNA polymerases alpha and delta in calf thymus DNA.

Most, although not all, samples of commercial calf thymus DNA were strongly inhibitory to DNA polymerase alpha; the inhibition made the DNA useless as a template for this enzyme. In a pre-assembled DNA polymerase assay mixture (minus enzyme but including activated DNA) the inhibition tended to diminish with time but at a rate that was not predictable, and some inhibition usually persisted. It was concluded that the inhibition was the result of contamination of the DNA by a heparin-like material on the basis of the following: 1) the inhibition could be reversed by treatment of the DNA with heparinase; 2) both the endogenous inhibitory effect of calf thymus DNA as well as the inhibitory effect of heparin on DNA polymerase alpha are reversed by protamine (which is known to prevent the antithrombin activity of heparin); 3) both the endogenous inhibition and inhibition by heparin are also reversed by ampholyte (which also prevents the antithrombin activity of heparin); and 4) both the endogenous and the heparin-induced inhibitory effects display the same spectrum of activity against mammalian DNA polymerases, i.e. both DNA polymerases alpha and delta are extremely sensitive whereas, DNA polymerases beta and gamma are resistant. The last result also suggests the use of heparin as a specific inhibitor of purified mammalian DNA polymerases alpha and delta, similar to the use of aphidicolin.     

Butylanilinouracil: a selective inhibitor of HeLa cell DNA synthesis and HeLa cell DNA polymerase alpha.

A series of 6-anilinouracils, dGTP analogues which selectively inhibit specific bacterial DNA polymerases, were examined for their capacity to inhibit purified DNA polymerases from HeLa cells. The p-n-butyl derivative (BuAU) was found to inhibit DNA polymerase alpha with a Ki of approximately 60 microM. The inhibitory effect of BuAU was reversed specifically by dGTP and was observed only for DNA polymerase alpha; polymerases beta and lambda were not inhibited by drug at concentrations as high as 1 mM. BuAU also was inhibitory in vivo in HeLa cell culture; at 100 microM it reversibly inhibited cell division and selectively depressed DNA synthesis. The results of these studies indicate that BuAU is an inhibitor with considerable potential as a specific probe with which to dissect the structure of mammalian polymerase alpha and its putative role in cellular DNA replication.     

Inhibitory effects of 5-alkyl- and 5-alkenyl-1-beta-D-arabinofuranosyluracil 5'-triphosphates on herpes virus-induced DNA polymerases

Various 5-substituted 1-beta-D-arabinofuranosyluracil 5'-triphosphates (H, methyl, ethyl, n-propyl, n-butyl, (E)-bromovinyl, styryl, and beta-phenylethyl derivatives) were prepared and their inhibitory effects on two different herpes virus-induced DNA polymerases (OMV and HCMV) were studied. These dTTP analogues inhibited the incorporation of [3H]dTMP into DNA in vitro. Among them, analogues having a vinyl group at the 5-position were strongly active against DNA polymerases induced on herpes virus infection. Kinetic analysis showed that the inhibition by the analogues was essentially competitive with respect to the substrate, dTTP. The K1 values (microM) for AraUTP (2.4), AraTTP (1.0), BVAUTP (0.8), and StUAUTP (0.8) were smaller than the Km value (microM) for dTTP (3.4), but those for AraEtUTP, AraPrUTP, and AraBuUTP (5-14) were larger than the Km for dTTP in the case of HCMV-induced DNA polymerase. In contrast to these results, OMV-induced DNA polymerase seemed to be more resistant to these inhibitors than HCMV-induced DNA polymerase. However, the mode of the structure of substituent groups at the 5-position of base moieties is almost the same for the two DNA polymerases, except for in the case of AraUTP itself.     

Substrate inhibitors of DNA biosynthesis

We present an account of the data on the inhibition analysis of DNA biosynthesis by substrate analogs with DNA polymerases of different origin. An attempt has been made to substantiate the factors that underline the specificity of inhibitors with respect to DNA synthesis that is catalyzed by various DNA polymerases.     

Protamine--a potent inhibitor of vesicular stomatitis virus transcriptase in vitro

The virion-associated RNA polymerase activity of vesicular stomatitis virus is inhibited by protamine at a concentration as low as 10^?7M. The inhibition is reversible, appears to be at the level of initiation and competitive with respect to ATP. Histone IIA and IV are also inhibitory whereas other fractions are not. The endogenous protein kinase activity is significantly inhibited by protamine. Virion-associated RNA or DNA polymerases of several animal viruses are also inhibited by protamine.     

Inhibition of the activities of DNA primase-polymerase alpha complex from KB cells by hexasodium sym-bis(m-aminobenzoyl-m-amino-p-methylbenzoyl-1-naphthylamino-4 ,6,8-trisulfonate)carbamide

A trypanocidal drug suramin [hexasodium sym-bis(m-amino-benzoyl-m-amino-p-methylbenzoyl-1-naphthylamino-4, 6, 8-trisulfonate)carbamide] was found to be a potent inhibitor of the activities of DNA primase and polymerase alpha from human KB cells. The mechanism of the inhibition by suramin was, however, quite different by these two polymerases. In the case of DNA primase, suramin inhibited competitively the incorporation of a nucleotide substrate, GTP, on the template polydeoxycytidylate, while the polymerase alpha was inhibited competitively by the drug with respect to the template primer (activated DNA). The observed inhibitory effect of suramin on nucleic acid synthesis seems to explain yet unknown mechanism of trypanocidal action of the drug.     

Programmed cell death of tobacco BY‐2 cells induced by still culture conditions is affected by the age of the culture under agitation

Evans Blue staining indicated that actively growing tobacco BY‐2 cells in the exponential phase died more rapidly than quiescent cells in the stationary phase when the cells cultured under agitation were placed under still conditions. Fifty percent cell death was induced at about 18, 26, 80 and 140 h for early, mid, late exponential‐ and stationary‐phase cells, respectively. Actively growing cells became TUNEL (transferase‐mediated dUTP nick end labelling)‐positive more rapidly than quiescent cells, suggesting that the cell death evaluated by Evans Blue is accompanied by DNA cleavages. Electrophoresis of genomic DNA showed a typical ‘DNA laddering' pattern formed by multiples of about 200 bp internucleosomal units. Chromatin condensation was first detected at least within 24 h by light microscopy, and then cell shrinkage followed. These findings suggest that the death of BY‐2 cells induced by still conditions is PCD (programmed cell death).     

Selective inhibition of DNA polymerase alpha by a polysaccharide purified from slime of Physarum polycephalum

A polysaccharide was purified from the slime of a myxomycete, Physarum polycephalum, and its inhibitory effect on eukaryotic DNA polymerases was examined. Almost all the calf thymus DNA polymerase alpha activity was inhibited with higher than 0.2 mg/ml of the polysaccharide, when the assay was carried out with activated DNA as a template. The inhibitory effect occurred regardless of the amounts of the enzyme and deoxyribonucleotides, however, kinetic analysis revealed that the inhibition occurs competitively with the template DNA, the Ki value being 4 micrograms/ml. Inhibition was observed for DNA polymerase alpha, but not for DNA polymerases beta and gamma from various eukaryote species.     

Cell cycle dependent activities of DNA polymerases alpha and delta in Chinese hamster ovary cells

The activities of DNA polymerases alpha and delta, in extracts from Chinese hamster ovary (CHO) cells, were assayed in order to determine whether these polymerases are regulated during the cell cycle. An exponential population of CHO cells was separated into enriched populations of G-1, S, and G-2/M phases of cell cycle by centrifugal elutriation. Total cell homogenates from each population were assayed for DNA polymerase activity by measuring labeled nucleotide incorporation into the exogenous templates oligo(dT).poly(dA) and DNase I activated calf thymus DNA. In these experiments, specific DNA polymerase inhibitors were added to assays of the cellular extracts to allow for the independent measurement of activities of DNA polymerases alpha and delta. Comparisons of total DNA polymerase activity from cellular extracts, sampled from each portion of the cell cycle, demonstrated no significant change with respect to the concentration of total protein. However, results indicate that the activity of DNA polymerase delta increases with respect to that of DNA polymerase alpha in the G-2/M portion of the cell cycle. This difference in relative activities of DNA polymerases alpha and delta suggests a coordinate regulation of a specific species of DNA polymerase during the cell cycle.     

Butylphenyl dGTP: a selective and potent inhibitor of mammalian DNA polymerase alpha.

BuPdGTP , the 2'-deoxyribonucleoside 5'-triphosphate of the DNA polymerase alpha (pol alpha)-specific inhibitor, N2-(p-n- butylphenyl )guanine, was examined with respect to its mechanism and its capacity to inhibit the mammalian DNA polymerases, pol alpha, pol beta, and pol gamma. BuP dGTP was specifically inhibitory for pol alpha, with no discernible activity on pol beta and pol gamma. The potency of BuP dGTP is unprecedented, with an apparent Ki less than 10 nanomolar. The unusual potency of the BuP dGTP is derived primarily from the 5' alpha and beta phosphoryl moieties, whose binding to enzyme complements that of the base-linked butylphenyl substituent. BuP dGTP is competitive with dGTP and apparently not subject to polymerization. Experiments employing BuP dGTP in the presence of a non-complementary template suggest that the core polymerase or an associated coprotein contains dNTP binding sites which recognize specific nucleic acid bases. The partial sensitivity of selected, non-mammalian DNA polymerases suggests that modification of the N2 substituent of dGTP will be a useful route to the design of novel, polymerase-specific affinity-probes.     

Uptake of l-Glutamate into Rat Brain Synaptic Vesicles: Effect of Inhibitors that Bind Specifically to the Glutamate Transporter

Abstract: In this study we have described a series of new and potent inhibitors of the vesicular uptake of glutamate. The two most efficient inhibitors were the dyes Evans blue and Chicago Skye Blue 6B, which are structurally related to glutamate and were competitive inhibitors in the nanomolar range. The anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (SITS) and the diuretics furosemide and bumetanide are inhibitors of chloride transport in other organs but were competitive inhibitors of glutamate and noncompetitive with respect to chloride ions. Evans blue, Chicago Skye Blue 6B, SITS, furosemide, and bumetanide are all large organic acids with two centers of negative charge and an electron-donating group at close vicinity of the negative charge at physiological pH. The inhibition of the glutamate uptake with these inhibitors was noncompetitive with respect to Cl⁻. The inhibitors, therefore, probably interact directly with the glutamate carrier. Bafilomycin A₁, which is a specific vacuolar ATPase inhibitor, was used as a control and inhibited the vesicular dopamine, glutamate, and GABA uptake to the same extent. None of the inhibitors had any effect on the plasma membrane carrier, which is therefore clearly different from the vesicular carrier.