DNA polymerase epsilon may be dispensable for SV40- but not cellular-DNA replication.

The contributions of DNA polymerases alpha, delta, and epsilon to SV40 and nuclear DNA syntheses were evaluated. Proteins were UV-crosslinked to nascent DNA within replicating chromosomes and the photolabelled polymerases were immunopurified. Only DNA polymerases alpha and delta were detectably photolabelled by nascent SV40 DNA, whether synthesized in soluble viral chromatin or within nuclei isolated from SV40-infected cells. In contrast, all three enzymes were photolabelled by the nascent cellular DNA. Mitogenic stimulation enhanced the photolabelling of the polymerases in the alpha>delta>epsilon order of preference. The data agree with the notion that DNA polymerases alpha and delta catalyse the principal DNA polymerisation reactions at the replication fork of SV40 and, perhaps, also of nuclear chromosomes. DNA polymerase epsilon, implicated by others as a cell-cycle checkpoint regulator sensing DNA replication lesions, may be dispensable for replication of the small, fast propagating virus that subverts cell cycle controls.

Assembly of simian virus 40 Okazaki pieces from DNA primers is reversibly arrested by ATP depletion.

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.

Cell-specific expression of immunoreactive cholesterol side-chain cleavage cytochrome P-450 during follicular development in the rat ovary.

Using a specific antiserum against rat cholesterol side-chain cleavage cytochrome P-450 (P-450scc), we examined the expression of this key steroidogenic enzyme during follicular development in PMSG-treated immature rats. The accumulation of the enzyme was monitored in ovary homogenates by quantitative immunodot blot assay, while expression of P-450scc in various cell types was visualized concomitantly by immunofluorescent staining of ovarian cryosections. Before PMSG treatment, no labeling of P-450scc could be observed in follicular granulosa cells. In contrast, steroidogenic cytochrome was markedly expressed in interstitial cells, part of theca interna cells, and hypertrophied theca of atretic follicles. As a result of PMSG treatment, the interstitial thecal cells promptly enriched their P-450scc content within 24 h, whereas the granulosa cells acquired the enzyme at a later time, between 30 and 48 h after hormone administration. After ovulation, many corpora lutea filled most of the ovarian volume, and the ovarian content of P-450scc was 47 times higher than that in control ovaries of untreated rats. In granulosa cell population of a single preovulatory follicle, a downward gradient of P-450scc expression was observed, starting high in the cells abutting the basal lamina and decreasing toward the cells lining the antrum. Cumulus cells failed to express P-450scc. Referring to the basal lamina, theca interna cells exhibited a reverse gradient of P-450scc expression, starting high in peripheral cells close to the theca externa layer and decreasing in cells located near the follicular basement membrane. Immunofluorescent labeling revealed a major difference between P-450scc expression in thecal cells compared to that in granulosa cells. While expression of P-450scc in granulosa cells was restricted exclusively to cells within preovulatory follicles, P-450scc labeling was observed throughout the ovary in thecal and interstitial cells associated with follicles at any phase of follicular maturation. Therefore, it may be proposed that the thecal and interstitial cells represent an all ovarian network which expresses its steroidogenic capacity at early stages of follicular maturation and thereby is able to supply androgens necessary for the follicular development.