Aphidicolin from

Klimczak et al. [139] have purified to homogeneity a monomeric DNA polymerase which is resistant to aphidicolin from the thermoacidophile S. acidocaldarius [ 2i9] and the methanogen M. thermoautotrophicum[ AO]. The DNA polymerase from... 

Deslongchamps and coworkers [26] used a combination of a transannular Diels-Alder cycloaddition and an intramolecular aldol reaction in the synthesis of the unnatural enantiomer of a derivative of the (+)-aphidicolin (4-74), which is a diterpe-noic tetraol isolated from the fungus Cephalosporium aphidicolia. This compound is an inhibitor of DNA polymerase, and is also known to act against the herpes simplex type I virus. In addition, it slows down eukaryotic cell proliferation, which makes it an interesting target as an anticancer agent... 

In the course of a total synthesis of aphidicolin (107), the conjugate addition of the dienoiate (104) to the chiral butenolide derivative (105) serves as a key step. A 7.4 1 mixture of diasteieomeric products is obtained, from which the major isomer (106) can be isolated in pure form after recrystaliization (Scheme 41).121 The selectivity of this remarkable reaction, in which two quaternary stereogenic centers are simultaneously generated in a highly selective manner, can be explained by the assumption that the reactants approach each other in the chelate-mode indicated in (108). 

Ring opening of the oxaspiropentane 343 upon treatment with sodium phenylselenide (vide supra, Sect. 4.5, Eq. (34)) 59) and O-silylation produce the vinylcyclopropanol trimethylsilyl ether 344 which, on flash thermolysis at 670 °C, gave the siloxycyclo-pentene 345 as a 2 1 mixture of epimers at C(8). Then, allylation of the more substituted enolate arising from 345, opens a convenient way to the antitumor agent, aphidicolin 346 181>. 

The partial synthesis from epicandicandiol of some C- and H-labelled kaurene derivatives has been described.These have possible application in the study of the biosynthesis of the Isodon diterpenoids. The syntheses of [17- C]kaur-16-en-20-ol from enmein and of 3-oxygenated derivatives of [17- C]kaur-16-ene from ent-3jS,19-dihydroxy-kaur-16-ene have also been described. The synthesis of radioactive aphidicolin has also been reported. It has been shown ° that ent-kaur-15-ene is formed by the dwarf mutant (ds) of maize in place of ent-kaur-16-ene. 

S. acidocaldarius, and more recently an aphidicolin-resistant DNA polymerase from... 

T. acidophilum, were purified to homogeneity in our laboratory [141,142]. Depending on the strain, the molecular mass of these monomeric DNA polymerases lies in the range of 70-100 kDa, which is close to the molecular mass of the aphidicolin-sensitive DNA polymerases isolated from other archaebacterial species. These enzymes are associated with a 3 to 5 exonuclease activity that could be involved in a proofreading mechanism (refs. [139,140,142], and our laboratory, unpublished results). In addition, the DNA polymerase from M thermoautotrophicum is associated with a 5 to 3 exonuclease activity [140], as is the case for eubacterial DNA polymerase I. In contrast, this activity has not been detected with DNA polymerases of thermoacidophiles. It has been shown that the 100 kDa DNA polymerase from S. acidocaldarius can be used in PCR [143,144]. 

An additional aphidicolin-resistant DNA polymerase composed of several 35 0 kDa polypeptides has been reported in S. acidocaldarius [146] and in T. acidophilum [ 41]. The authors have proposed that this DNA polymerase could be homologous to the eukaryotic DNA polymerase (3, which is a monomeric enzyme composed of a 40 kDa polypeptide. However, we have shown that in each case, the 35-40 kDa polypeptides are devoid of DNA polymerase activity when they are totally separated from the high-molecular-mass DNA polymerase already described[141,142],... 

In the halophile H. halobium, Nakayama et al. [148] have described an aphidicolin-sensitive DNA polymerase that they named DNA polymerase a . The enzyme has a high sedimentation coefficient value, is able to synthesize RNA on a synthetic DNA template, and is associated with a 3 to 5 exonuclease activity. Two major polypeptides of 60 and 70kDa were detected in a purified fraction of H. halobium DNA polymerase a [149]. The authors concluded from these results that this enzyme corresponds to a multi-subunit DNA polymerase able to perform DNA priming like eukaryotic DNA polymerase a. However, the association of the 60 and 70 kDa polypeptides with the polymerase activity has not been demonstrated and the specific activity of the enzyme is very low compared to any other purified DNA polymerases. 

The report of two DNA polymerases with different sensitivity to aphidicolin in the same species is strikingly different from the situation observed in methanogens and sulfothermophiles. Furthermore, the structure and size of the aphidicolin-sensitive DNA polymerase from H. halobium depart from those of the monomeric aphidicolin-sensitive DNA polymerases isolated in other archaebacteria. The reason for such discrepancies is not clear at the moment. 

Table 4 summarizes the properties of the different DNA polymerases which have been purified from archaebacteria. It is clear from studies performed in vivo that halophiles and some methanogens have at least one aphidicolin-sensitive DNA polymerase involved in DNA replication (most probably a replicase) on the other hand, only one DNA polymerase, either sensitive or resistant to aphidicolin, has been detected in various archaebacteria (with the exception of H. halobium). What are the phylogenetic relationships between these aphidicolin-resistant and -sensitive enzymes In collaboration with F. Lottspeich, we obtained the amino-acid sequences of several peptides from S. acidocaldarius DNA polymerase (resistant) and we observed that these sequences are present in the primary structure of S. solfataricus DNA polymerase (sensitive). This suggests that aphidicolin-resistant and aphidicolin-sensitive DNA polymerases detected in archaebacteria are homologous. It remains to determine whether aphidicolin-resistant enzymes contain the amino-acid motifs typical for DNA polymerases of the B family. 

Although the data on the archaebacterial DNA world are far from exhaustive, it is nevertheless already possible to draw tentative phylogenetic considerations. In their transcription and translation machineries, archaebacteria exhibit several eukaryotic-like features, compared to those in eubacteria, such as an RNA polymerase and elongation factors of the eukaryotic type (see ref. [151], and other chapters of this volume). Several features of the DNA world have been frequently considered to be eukaryotic the presence of histone-like proteins, first HTa[152], more recently HMf[23], the sensitivity of halobacteria to drugs otherwise specific of the eukaryotic DNA topoisomerasell [103], and the existence of an aphidicolin-sensitive DNA polymerase [124]. However, it appears that HTa is a close relative of eubacterial HU proteins (see Fig. 2), that Haloferax Type II DNA topoisomerase is very similar to eubacterial DNA gyrases (see Fig. 9) and that aphidicolin also inhibits an eubacterial DNA polymerase. HMf is clearly homologous to eukaryotic histones, but HMf nucleosomes are drastically different from eukaryotic ones furthermore, one cannot exclude the existence of HMf-related proteins in eubacteria. 

Cell growth during the time of the assay might influence the results, in the sense that the filling of the artificial wound could be contributed from both cell replication and movement. In order to avoid this problem, the assay should be conducted in serum-free or at least low-serum conditions, to limit cell growth. Alternatively, inhibitors of DNA synthesis (mitomycin-C or aphidicolin at 0.5 /Lig/ml), can be included in the culture medium, since it has been shown that active migration may depend on protein, but not DNA synthesis (Geimer and Bade, 1991 Chen et al., 1994). 

Non-dNTP inhibitors aphidicolin (10 mg/mL in water), phosphonoformic acid (10 mg/mL in water), actinomycin D (10 mg/mL in ethanol). Store frozen, protected from light, in small aliquots. Make up 400 pg/mL solutions just before use. 

Figure 3. Role of enhancers and histone acetylation in regulating gene expression. A plasmid-borne reporter gene was either driven by the tk promoter (Promoter) or was coupled with the FI 01 enhancer (Enhancer). In some cases the embryos were also cultured in the presence of butyrate, which induces histone hyperacetylation (Promoter-FButyrate, Enhancer-FButyrate). Either male pronucleus (Male PN), female pronucleus (Female PN), or zygotic nucleus (two-cell) was injected. When one-cell embryos were injected, they were cultured in the presence of aphidicolin to a time that corresponded to the mid to late Iwo-cell stage. The data are expressed relative to the amount of luciferase activity detected in the male pronucleus of an S-phase-arrested, one-cell embryo and were taken from Wiekowski et al., 1993.

Figure 5. Role of DNA replication in the development of a transcriptionally repressive state. BrUTP incorporation of membrane-permeabilized embryos was conducted on two-cell embryos in G2 (2C) or S-phase-arrested, one-cell embryos that were analyzed at a time that corresponded to G2 of the two-cell stage. Both the male (aph M) and female (aph F) were analyzed. Transcription was also analyzed at a time that corresponded to C2 of the two-cell stage for S-phase-arrested, two-cell embryos (aphidicolin added after the first round of replication aph 2C) or in two-cell embryos treated with trapoxin (trap). The data are expressed relative to the amount of BrUTP incorporated by a two-cell blastomere nucleus in C2. (Data taken from Aoki et al., 1997).

The biosynthesis of aphidicolin has been examined (Scheme 5.11). The enrichment and coupling patterns of aphidicolin biosynthesized from [l- C]-, [2- C]- and [l,2- C2]acetate were used to define the constituent isoprene units. The numbers of 2- and 5- mevalonoid hydrogens that were incorporated were established by tritium labelling. The generation of a coupling, which... 

Biosynthesis of the diterpenoid aphidicolin. Isolation of intermediates from P450 inhibitor treated mycelia of Phoma betae, H. Oikawa, S. Ohashi, A. Ichihara and S. Sakamura, Tetrahedron, 1999, 55, 7541. 

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