Mechanism of DNA polymerase .alpha. inhibition by aphidicolin

Sheaff, Robert J.; Ilsley, Diane; Kuchta, Robert D.

doi:10.1021/bi00099a014

Cited by 97 publications

(92 citation statements)

References 30 publications

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“…This premature termination of primer extension may be due to thiocoraline-induced dissociation of DNA polymerase α from the elongating primer/template or, alternatively, 'trapping' the enzyme, presumably due to the formation of an enzyme-thiocoraline-DNA ternary complex. The latter mechanism has been proposed to explain aphidicolininduced inhibition of DNA synthesis (Sheaff et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

Mode of action of thiocoraline, a natural marine compound with anti-tumour activity

et al. 1999

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Thiocoraline, a new anticancer agent derived from the marine actinomycete Micromonospora marina , was found to induce profound perturbations of the cell cycle. On both LoVo and SW620 human colon cancer cell lines, thiocoraline caused an arrest in G1 phase of the cell cycle and a decrease in the rate of S phase progression towards G2/M phases, as assessed by using bromodeoxyuridine/DNA biparametric flow cytometric analysis. Thiocoraline does not inhibit DNA-topoisomerase II enzymes in vitro, nor does it induce DNA breakage in cells exposed to effective drug concentrations. The cell cycle effects observed after exposure to thiocoraline appear related to the inhibition of DNA replication. By using a primer extension assay it was found that thiocoraline inhibited DNA elongation by DNA polymerase α at concentrations that inhibited cell cycle progression and clonogenicity. These studies indicate that the new anticancer drug thiocoraline probably acts by inhibiting DNA polymerase α activity. © 1999 Cancer Research Campaign

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Section: Discussionmentioning

confidence: 99%

Mode of action of thiocoraline, a natural marine compound with anti-tumour activity

et al. 1999

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“…These studies attempted to classify DNA polymerases as eukaryotic-or eubacterial-like on the basis of enzymatic properties and resistance or sensitivity to various inhibitors. The most common indicator of the presence of a eukaryotic-like DNA polymerase was sensitivity to aphidicolin, a fungal metabolite which inhibits eukaryotic DNA replication by allosterically binding to the replicative DNA polymerases (45). By using aphidicolin sensitivity as an indicator, S. acidocaldarius and S. solfataricus were found to possess a eukaryoticlike DNA polymerase activity as well as an unclassified aphidicolin-resistant DNA polymerase activity (14,29,35,41,49).…”

Section: Discussionmentioning

confidence: 99%

Gene duplications in evolution of archaeal family B DNA polymerases

1997

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All archaeal DNA-dependent DNA polymerases sequenced to date are homologous to family B DNA polymerases from eukaryotes and eubacteria. Presently, representatives of the euryarchaeote division of archaea appear to have a single family B DNA polymerase, whereas two crenarchaeotes, Pyrodictium occultum and Sulfolobus solfataricus, each possess two family B DNA polymerases. We have found the gene for yet a third family B DNA polymerase, designated B3, in the crenarchaeote S. solfataricus P2. The encoded protein is highly divergent at the amino acid level from the previously characterized family B polymerases in S. solfataricus P2 and contains a number of nonconserved amino acid substitutions in catalytic domains. We have cloned and sequenced the ortholog of this gene from the closely related Sulfolobus shibatae. It is also highly divergent from other archaeal family B DNA polymerases and, surprisingly, from the S. solfataricus B3 ortholog. Phylogenetic analysis using all available archaeal family B DNA polymerases suggests that the S. solfataricus P2 B3 and S. shibatae B3 paralogs are related to one of the two DNA polymerases of P. occultum. These sequences are members of a group which includes all euryarchaeote family B homologs, while the remaining crenarchaeote sequences form another distinct group. Archaeal family B DNA polymerases together constitute a monophyletic subfamily whose evolution has been characterized by a number of gene duplication events.Studies on the mechanisms of DNA replication in eubacteria and eukaryotes have led to the identification of numerous DNA-dependent DNA polymerases (31). These have been classified into families based on amino acid sequence similarity of the catalytic subunit to one of the three Escherichia coli DNA polymerases (5). Family A DNA polymerases include E. coli DNA polymerase I (polI), all eubacterial polI homologs, some eubacterial phage DNA polymerases, and mitochondrial DNA polymerases (often called ␥ polymerase). Family B DNA polymerases include E. coli DNA polymerase II, some eubacterial phage DNA polymerases, the eukaryotic nuclear replicative DNA polymerases (␣, ␦, and ε), and eukaryotic viral and plasmid-borne enzymes. Family C includes only eubacterial polIII homologs: there are no known phage, viral, archaeal, or eukaryotic family C DNA polymerases. An additional eukaryotic nuclear encoded DNA polymerase, ␤, which functions in repair, is assigned to family X. Members of family X have little amino acid sequence similarity with DNA polymerases but instead exhibit amino acid sequence similarity to terminal transferases.Although our understanding of DNA replication in eubacteria and eukaryotes is quite advanced, comparatively little is known about DNA replication in archaea. Early studies on DNA replication showed that aphidicolin, a specific inhibitor of eukaryotic DNA replication, inhibited cell growth and DNA synthesis in halophilic archaea (21, 43). Aphidicolin-sensitive DNA polymerases were subsequently purified from halophilic, methanogenic, and some thermo...

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“…Aphidicolin is an inhibitor of replicative DNA polymerases and inhibits DNA synthesis without a corresponding decrease in deoxynucleotides. 25 At these concentrations, growth inhibition and DNA synthesis inhibition are similar to HU. When incubated with both aphidicolin and GCV for 24 h, bystander cells were actually protected from the cytotoxic effect of GCV.…”

Section: In Vitro Gcv Cytotoxicity With Hu and Aphidicolinmentioning

confidence: 89%