Cyclin-dependent kinase inhibits reinitiation of a normal S-phase program during G2 in fission yeast

Abstract

To achieve faithful replication of the genome once in each cell cycle, reinitiation of S phase is prevented in G(2) and origins are restricted from refiring within S phase. We have investigated the block to rereplication during G(2) in fission yeast. The DNA synthesis that occurs when G(2)/M cyclin-dependent kinase (CDK) activity is depleted has been assumed to be repeated rounds of S phase without mitosis, but this has not been demonstrated to be the case. We show here that on G(2)/M CDK depletion in G(2), repeated S phases are induced, which are correlated with normal G(1)/S transcription and attainment of doublings in cell size. Mostly normal mitotic S-phase origins are utilized, although at different efficiencies, and replication is essentially equal across the genome. We conclude that CDK inhibits reinitiation of S phase during G(2), and if G(2)/M CDK is depleted, replication results from induction of a largely normal S-phase program with only small differences in origin usage and efficiency.

FIG. 1.

Single-cell methods for studying DNA synthesis and MBF-dependent gene expression. (A) BrdU pulse-labeling of wild-type and wee1-50 cells. Left panels: a leu1-32 his7-366 pJL218 (his7 adh1-tk) pFS181 (leu1 adh1-hENT1) culture at 32°C was pulse-labeled with BrdU. BrdU was detected by immunofluorescence and DNA visualized by 4′,6′-diamidino-2-phenylindole (DAPI) staining. The scale bar is 5 μm long. The percentage of BrdU-positive nuclei was plotted as a function of fixed cell volume per nucleus. Right panel: a wee1-50 leu1-32 his7-366 pJL218 (his7 adh1-tk) pFS181 (leu1 adh1-hENT1) culture at 25°C was shifted to 32°C for 6 h. Cells were pulse-labeled with BrdU. (B) MBF-dependent gene expression in wild-type and wee1-50 cells. The cell volume per nucleus with peak Tos4-GFP signal was determined. Left panels: an ade6-M210 tos4-GFP::KanMx6 culture at 32°C. The percentage of nuclei with a strong GFP signal was plotted as a function of cell volume per nucleus. Right panel: a wee1-50 ade6-M210 tos4-GFP::KanMx6 culture at 25°C was shifted to 32°C for 6 h. The percentage of nuclei with a strong Tos4-GFP signal was plotted as a function of cell volume per nucleus. (C) FACS analysis of wild-type cells at 32°C and wee1-50 cells shifted from 25°C to 32°C for 6 h. Wee1-50 cells show a 1C peak. (D) Experiment schematic: a cdc25-22 ade6-M210 tos4-GFP::KanMx6 culture at 25°C was shifted to 36.5°C for 4 h and released at 25°C. (E) FACS analysis: cdc25-22 tos4-GFP cycled similarly to cdc25-22 alone. (F) The percentage of nuclei with strong Tos4-GFP signal was plotted as a function of time, peaking at the end of S phase. Tos4-GFP localizes to the nucleus periodically during the cell cycle. (G) A cycling cdc10-v50 Δcdc13::ura4⁺ pREP45 cdc13⁺ ade6-704 tos4-GFP::KanMx6 culture in the absence of thiamine at 25°C was shifted to 36.5°C for 4 h. The percentage of nuclei in the culture with a strong Tos4-GFP signal was assessed every hour. (H) Rereplication was induced in a cdc10-v50 Δcdc13::ura4⁺ pREP45 cdc13⁺ ade6-704 tos4-GFP::KanMx6 cul- ture. At 6 h after refeeding nitrogen, half of the culture was shifted to 36.5°C for 3 h (gray line, block) while half continued to rereplicate at a permissive temperature (dashed line, control). At 9 h, half of the blocked culture was released to 25°C (black line, block release). The percentage of nuclei with Tos4-GFP was determined at each time point.

FIG. 2.

Periods of DNA synthesis correspond to cell volume doublings in cdc13 switch-off. (A) BrdU pulse-labeling of cells lacking Cdc13. An exponentially growing Δcdc13::ura4⁺ pREP45 cdc13⁺ ade6-704 leu1 adh1-hENT his adh1-tk culture was filtered and resuspended in medium lacking nitrogen. After 8 h, thiamine was added, and after an additional 12 h, nitrogen was replenished and the culture shifted to 32°C. Cells depleted of the Cdc13 cyclin were pulse-labeled with BrdU during a time course as cells underwent DNA doublings from 1C to 32C. DNA was visualized by 4′,6′-diamidino-2-phenylindole (DAPI) staining, and BrdU was detected by indirect immunofluorescence. Cells from 13 h after replenishment of nitrogen are shown. The scale bar is 5 μm long. (B) The percentage of BrdU-positive cells was plotted as a function of time. (C) Cells from all time points were binned according to fixed cell volume, and the percentage of cells with incorporated BrdU in each bin was plotted as a function of cell volume. Peaks in the percentage of BrdU-positive cells corresponded to DNA doublings.

FIG. 3.

MBF-dependent gene expression is periodic in cdc13 switch-off. Nuclear signal of Tos4-GFP in the absence of cdc13. Left panel: Δcdc13::ura4⁺ pREP45 cdc13⁺ ade6-704 tos4-GFP::KanMx6 cells depleted of Cdc13. DNA content increased to 16C. Right panel: the percentage of cells with strong nuclear GFP as a function of cell volume.

FIG. 4.

Equal replication across the genome in a cdc13 switch-off strain. DNA from rereplicating cells which had attained 16C and 32C DNA contents in the absence of HU was hybridized against a reference to microarrays. Signal was normalized to the rest of the genome.

FIG. 5.

Replication origin usage in a cdc13 cyclin switch-off. Mapping origins in endoreduplication of a cdc13 cyclin switch-off mutant. (A) Left panel: schematic of the cdc13 cyclin switch-off time course experiment. Cells were nitrogen starved for 5 h at 25°C in the presence of thiamine. Nitrogen was replenished and the culture shifted to 32°C, and 11 mM HU was added. Right panel: FACS analysis of the cdc13 switch-off mutant, induced into endoreduplication with or without the addition of 11 mM HU. In the presence of HU, cells block at the onset of the first round of endoreduplication. In the absence of HU, cells undergo repeated rounds of replication without intervening mitoses. (B) Comparison of wild-type and cdc13 switch-off replication profiles from the average for two HU experiments. Examples of origins: arrows mark origins which are used both in a normal S phase and in the cdc13 s/o strain, gray circles mark normal S-phase origins not used in cdc13 s/o, and asterisks mark new origins which are used only in the cdc13 s/o strain. The regions were selected for illustration because they contain all three types of origins.