aphidicolin and 2--deoxycytidine-5--triphosphate

Werner syndrome is a genetic disorder characterized by premature aging and cancer-prone symptoms, and is caused by mutation of the WRN gene. WRN is a member of the RecQ helicase family and is thought to function in processes implicated in DNA replication and repair to maintain genome stability; however, its precise function is still unclear. We found that replication fork arrest markedly enhances chromatin binding of focus-forming activity 1 (FFA-1), a Xenopus WRN homolog, in Xenopus egg extracts. In addition to FFA-1, DNA polymerase delta (Poldelta) and replication protein A, but not DNA polymerase epsilon and proliferating cell nuclear antigen, accumulated increasingly on replication-arrested chromatin. Elevated accumulation of these proteins was dependent on formation of pre-replicative complexes (pre-RCs). Double-strand break (DSB) formation also enhanced chromatin binding of FFA-1, but not Poldelta, independently of pre-RC formation. In contrast to FFA-1, chromatin binding of Xenopus Bloom syndrome helicase (xBLM) only slightly increased after replication arrest or DSB formation. Thus, WRN-specific, distinct processes can be reproduced in the in vitro system in egg extracts, and this system is useful for biochemical analysis of WRN functions during DNA metabolism.

Topics: Adenosine Triphosphatases; Animals; Aphidicolin; Cell Cycle Proteins; Chromatin; Deoxycytosine Nucleotides; DNA Damage; DNA Helicases; DNA Polymerase II; DNA Polymerase III; DNA Replication; DNA-Binding Proteins; Female; Geminin; Male; Oocytes; Proliferating Cell Nuclear Antigen; RecQ Helicases; Replication Protein A; Spermatozoa; Werner Syndrome Helicase; Xenopus; Xenopus Proteins

A variety of isosteres of the DNA polymerase inhibitor aphidicolin were synthesized as potential antiherpes agents. Modeling studies indicated that the bicyclooctane C, D rings of aphidicolin could be replaced by an aromatic moiety while maintaining the spatial arrangement of the hydroxyl group equivalent to the essential C18 hydroxyl group of aphidicolin. Of the racemic isosteres synthesized only 13, the compound with the greatest structural similarity to aphidicolin, showed any significant antiviral activity in primary assays. An enantioselective synthesis of the compound was carried out and the 4aS isomer 36 was shown to account for the observed antiviral activity noted against herpes simplex virus 1 and human cytomegalovirus.

Topics: Antiviral Agents; Aphidicolin; Binding Sites; Crystallography, X-Ray; Cytomegalovirus; Deoxycytosine Nucleotides; Herpesvirus 1, Human; Herpesvirus 2, Human; Models, Molecular; Molecular Structure; Nucleic Acid Synthesis Inhibitors; Phenanthrenes; Stereoisomerism; Structure-Activity Relationship

We have previously proposed that DNA polymerase alpha-primase provides short RNA-DNA precursors below 40 nucleotides (DNA primers), several of which assemble into an Okazaki piece after intervening RNA has been removed and the gaps have been filled by DNA polymerase delta (or epsilon) (T. Nethanel, S. Reisfeld, G. Dinter-Gottlieb, and G. Kaufmann, J. Virol. 62:2867-2873, 1988; T. Nethanel and G. Kaufmann, J. Virol. 64:5912-5918, 1990). In this report, we confirm and extend these conclusions by studying the effects of deoxynucleoside triphosphate (dNTP) concentrations and the presence of ATP on the occurrence, dynamics, and configuration of DNA primers in simian virus 40 replicative intermediate DNA. We first show that these parameters are not significantly affected by a 10-fold increase in dNTP precursor concentrations. We then demonstrate that Okazaki piece synthesis can be arrested at the level of DNA primers by ATP depletion. The arrested DNA primers faced short gaps of 10 to 20 nucleotides at their 3' ends and were progressively chased into Okazaki pieces when ATP was restored. ATP could not be substituted in this process by adenosine-5'-O-(3-thiotriphosphate) or adenyl-imidodiphosphate. The chase was interrupted by aphidicolin but not by butylphenyl-dGTP. The results implicate an ATP-requiring factor in the switch between the two DNA polymerases engaged in Okazaki piece synthesis. They also suggest that the replication fork advances by small, DNA primer-size increments.

Topics: Adenosine Triphosphate; Aphidicolin; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyuracil Nucleotides; DNA; DNA Primase; DNA Replication; DNA, Single-Stranded; DNA, Viral; Nucleic Acid Conformation; RNA Nucleotidyltransferases; Simian virus 40

17-Acetylaphidicolin was 10-fold weaker and two derivatives lacking hydroxyl groups at the 16 and 17 positions were 100-fold weaker than aphidicolin as inhibitors of DNA polymerase alpha from HeLa and Chinese hamster ovary cells. 17,18-Diacetyl, 3,17,18-triacetyl and 3-epi derivatives of aphidicolin were inactive. Active compounds were, like aphidicolin, competitive with dCTP and did not inhibit aphidicolin-resistant DNA polymerases.

Topics: Animals; Aphidicolin; Cricetinae; Cricetulus; Deoxycytosine Nucleotides; Diterpenes; DNA Polymerase II; Female; Fibroblasts; HeLa Cells; Ovary

In connection with the characterization of two DNA polymerases (DPols) of Chlorella, we have extensively surveyed the literature on inhibition studies on DPols in various eukaryotes. By applying Tamiya's plot (1), we have obtained two parameters for each of the inhibitors, phi- and n-values, which express the enzyme sensitivity to the drug and the number of inhibitor molecules present in the enzyme-inhibitor complex that is principally involved in the inhibition, respectively. By inspecting these parameters for the three mammalian DPols, alpha-, beta-, and gamma-pols, as well as other eukaryotic DPols, we have found that: [1] inhibitors commonly utilized for characterizing various DPols can be classified into two major groups, each having two subgroups, on the basis of a comparison of the phi values among alpha-, beta-, and gamma-pols. Moreover, the grouping seems not to be merely coincidental, but to be intrinsically related to facets of the enzyme reaction, which may be taken to reflect evolutionary differences in DPol structure and function among the three DPols; [2] the remarkable n value, n = 1/2, that has been found for the inhibitors competitive with dCTP in Chlorella DPols has also been detected widely in many other eukaryotic DPols. Based on the first finding as well as many other data on various DPols, we have proposed an evolutionary scenario for eukaryotic DPols. Based on the second finding, we have hypothesized a novel role for dCTP as a cofactor, probably an apparent allosteric effector, in the nucleotide transfer reaction mechanism.

Topics: Aphidicolin; Arabinofuranosylcytosine Triphosphate; Biological Evolution; Cations, Divalent; Chlorophyta; Deoxycytosine Nucleotides; Dideoxynucleotides; Diphosphates; Diterpenes; Ethidium; Ethylmaleimide; Nucleic Acid Synthesis Inhibitors; Thymine Nucleotides

Topics: Animals; Aphidicolin; Cell Line; Cell Survival; Cricetinae; Deoxycytosine Nucleotides; Deoxyribonucleotides; Diterpenes; DNA; DNA Polymerase II; Drug Resistance; Female; Methylnitronitrosoguanidine; Mutation; Ovary; Thymine Nucleotides; Ultraviolet Rays

Aphidicolin, a mycotoxin that inhibits eucaryotic DNA polymerase alpha, blocked the growth of Toxoplasma gondii in confluent cultured human fibroblasts. Aphidicolin immediately inhibited DNA synthesis by T. gondii while it had a delayed and less dramatic effect on RNA synthesis. A mutant of T. gondii resistant to aphidicolin was isolated with the aid of mutagenesis by ethylnitrosourea. Parasite growth measured three days after drug treatment and parasite DNA synthesis measured immediately after drug treatment were, respectively, five- and four-fold more resistant to aphidicolin in the mutant as compared with the wild type parasite. The mutant had a three-fold greater capacity than the wild type to incorporate uracil into its deoxycytidine triphosphate pool. This increased deoxycytidine triphosphate pool is the probable explanation for the mutant's resistance because this deoxynucleotide is known, in mammalian cells, to reverse the inhibition of DNA synthesis by aphidicolin in a competitive manner.

Topics: Animals; Aphidicolin; Deoxycytosine Nucleotides; Diterpenes; DNA; Drug Resistance, Microbial; Mutation; RNA; Toxoplasma; Uracil

In nongrowing mammalian cells, DNA repair synthesis following irradiation with high doses of UV is almost totally inhibited by aphidicolin, an agent specific for DNA polymerase alpha, and presumably is mediated by that polymerase. In this paper, several enzymologic characteristics of DNA repair synthesis induced in permeable confluent diploid human fibroblasts by high doses of UV have been examined and compared with corresponding features of semiconservative DNA replication, a process which is also mediated by polymerase alpha. Inhibition of UV-induced repair synthesis required doses of aphidicolin about 20-fold higher than those needed to inhibit replication, even when the two processes were studied at identical salt and nucleotide concentrations. As is the case for replication, inhibition of UV-induced repair synthesis by aphidicolin is competitive with dCTP. The apparent Ki values for aphidicolin of the two processes are similar, 0.2 microM for repair synthesis and 0.1 microM for semiconservative replication. In contrast, the apparent Km values for dCTP are very different, 0.17 microM for repair synthesis and about 2 microM for replication. The apparent Km values for all four deoxyribonucleoside triphosphates varied together are also very different, 0.07 microM for repair and 30 microM for replication. These results suggest that either UV-induced DNA repair synthesis and semiconservative replication are mediated by two different aphidicolin-sensitive DNA polymerases or the two functions are performed by a single polymerase (e.g. polymerase alpha) which, as a result of accessory proteins or other factors, acquires very different enzymologic characteristics.

Topics: Aphidicolin; Deoxycytosine Nucleotides; Diterpenes; DNA Polymerase II; DNA Repair; DNA Replication; Fibroblasts; Humans; Kinetics; Time Factors; Ultraviolet Rays

Topics: Animals; Aphidicolin; Binding, Competitive; Cell Division; Deoxycytosine Nucleotides; Diterpenes; DNA; DNA Polymerase II; DNA Replication; HeLa Cells; Plants

dNTP pools are quite low in immature oocytes of the starfish, expand during the 1-methyladenine-induced maturational process and thereafter reach a maximal level (approx. 35, 20, 15 and 5 fmoles/egg for dTTP, dCTP, dATP and dGTP, respectively) which is maintained in overmatured eggs. Maturing oocytes were inseminated at the stage just before extrusion of the first polar body and determination of dNTP pools during early embryogenesis showed the same expansion pattern as that of the 1-methyladenine-treated oocytes. Therefore, the increase in dNTP pools during early embryogenesis is dependent on 1-methyladenine (1-MA) but independent of fertilization. Aphidicolin, a specific inhibitor of eukaryotic DNA polymerase alpha, has no effect on dNTP pool size in 1-methyladenine-treated oocytes, but causes considerable expansion of dNTP pools in fertilized eggs which cleave achromosomally in the presence of the drug.

Topics: Animals; Aphidicolin; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Diterpenes; Female; Oocytes; Oogenesis; Ovum; Starfish; Thymine Nucleotides; Zygote