The Mode of Inhibitory Action by Aphidicolin on Eukaryotic DNA Polymerase α

Oguro, Mieko; Suzuki-Hori, Chiyo; Nagano, Hiroshi; Mano, Yoshitake; Ikegami, Susumu

doi:10.1111/j.1432-1033.1979.tb13149.x

Cited by 142 publications

(71 citation statements)

References 17 publications

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“…Both DNA polymerase a (21) and cellular DNA synthesis in vitro show an increased sensitivity at low dCTP concentrations, which are close to the intracellular concentration of dCTP in vivo. At these low concentrations a good correlation exists between the sensitivity of DNA polymerase a and DNA synthesis in isolated nuclei or intact cells.…”

Section: Resultsmentioning

confidence: 99%

Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis

Kwant

Vliet

1980

Nucl Acids Res

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There is strong evidence for a participation of DNA polymerase y in the replication of adenovirus (Ad) DNA. To study a possible additional role of DNA polymerase a we measured the effect of aphidicolin on viral DNA replication.In intact cells, aphidicolin inhibits Ad DNA synthesis weakly. The drug concentration required for 50% inhibition of Ad DNA replication was 300-400 fold higher than for a similar effect on cellular DNA synthesis. Such a differential inhibition was also observed in AGMK cells doubly infected with SV40 and the simian adenovirus SA7. No evidence was found for modification of aphidicolin in infected cells or for a change in aphidicolin sensitivity of DNA polymerase a after infection.The extent of inhibition of purified DNA polymerase a was dependent upon the dCTP concentration. The same situation was observed when DNA synthesis was studied in isolated nuclei from uninfected cells. However, in nuclei from Ad infected cells no effect of dCTP on aphidicolin sensitivity was found. These results were taken as evidence that DNA polymerase a does not participate in the replication of adenovirus DNA.

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Section: Resultsmentioning

confidence: 99%

Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis

Kwant

Vliet

1980

Nucl Acids Res

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“…The finding that cdc2 phosphorylates eEF2K at Ser359 could therefore be important for the control of eEF2K, eEF2 and translation elongation during M-phase. To study this further, HeLa cells were blocked in early S-phase using the DNA polymerase inhibitor aphidicolin (Oguro et al, 1979). At various times after release from this block, progress through the cell cycle was monitored by flow cytometry ( Figure 5A).…”

Section: Regulation Of Eef2k and Eef2 Phosphorylation During The Cellmentioning

confidence: 99%

cdc2–cyclin B regulates eEF2 kinase activity in a cell cycle- and amino acid-dependent manner

Smith

Proud

2008

EMBO J

101

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The calcium/calmodulin‐dependent kinase that phosphorylates and inactivates eukaryotic elongation factor 2 (eEF2 kinase; eEF2K) is subject to multisite phosphorylation, which regulates its activity. Phosphorylation at Ser359 inhibits eEF2K activity even at high calcium concentrations. To identify the kinase that phosphorylates Ser359 in eEF2K, we developed an extensive purification protocol. Tryptic mass fingerprint analysis identified it as cdc2 (cyclin‐dependent kinase 1). cdc2 co‐purifies with Ser359 kinase activity and cdc2–cyclin B complexes phosphorylate eEF2K at Ser359. We demonstrate that cdc2 contributes to controlling eEF2 phosphorylation in cells. cdc2 is activated early in mitosis. Kinase activity against Ser359 in eEF2K also peaks at this stage of the cell cycle and eEF2 phosphorylation is low in mitotic cells. Inactivation of eEF2K by cdc2 may serve to keep eEF2 active during mitosis (where calcium levels rise) and thereby permit protein synthesis to proceed in mitotic cells. Amino‐acid starvation decreases cdc2's activity against eEF2K, whereas loss of TSC2 (a negative regulator of mammalian target of rapamycin complex 1(mTORC1)) increases it. These data closely match the control of Ser359 phosphorylation and indicate that cdc2 may be regulated by mTORC1.

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“…Accumulation of early RI DNA in the presence of aphidicolin appeared to result from the action of this drug on DNA polymerase a. Inhibition of DNA polymerase a by aphidicolin involves competition with dCTP (20). Addition Fig.…”

mentioning

confidence: 99%

Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

Decker¹,

Yamaguchi²,

Possenti³

et al. 1986

Mol. Cell. Biol.

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Aphidicolin, a specific inhibitor of DNA polymerase a, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (on) and continuation of replication beyond on. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600-to 700-base-pair region centered at on. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 on-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the on region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of on, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase a. Synthesis of pppA/G(pN)64(pdN)21_23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at on first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.Simian virus 40 (SV40) is a small (5.2 kilobases [kb]) DNA virus that replicates in the nuclei of permissive monkey or human cells as a circular chromosome whose nucleosome striicture and histone composition are indistinguishable from that of its host (reviewed in reference 5 and in M. L. DePamphilis and M. K. Bradley, in N. P. Salzman, ed., The Viruses: Polyoma Viruses, in press). With the exception of initiation of viral DNA replication, all steps in the replication and assembly of viral chromosomes are carried out exclusively by cellular components. Initiation of SV40 DNA replication requires a cis-acting sequence that functions as the origin of replication (ori), SV40 large tumor antigen (T-ag), and one or more permissive cell factors. Bidirectional replication then begins at the junction between the strongest DNA-binding site for T-ag and the ori core. Analysis of initiation sites for RNA-primed DNA synthesis in SV40 replicating intermediates led to the hypothesis that initiation of DNA synthesis at ori involves the same mechanism used to synthesize Okazaki...

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The Mode of Inhibitory Action by Aphidicolin on Eukaryotic DNA Polymerase α

Cited by 142 publications

References 17 publications

Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis

Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis

cdc2–cyclin B regulates eEF2 kinase activity in a cell cycle- and amino acid-dependent manner

Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

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