The role and regulation of the preRC component Cdc6 in the initiation of premeiotic DNA replication

Abstract

In all eukaryotes, the initiation of DNA replication is regulated by the ordered assembly of DNA/protein complexes on origins of DNA replication. In this report, we examine the role of Cdc6, a component of the prereplication complex, in the initiation of premeiotic DNA replication in budding yeast. We show that in the meiotic cycle, Cdc6 is required for DNA synthesis and sporulation. Moreover, similarly to the regulation in the mitotic cell cycle, Cdc6 is specifically degraded upon entry into the meiotic S phase. By contrast, chromatin-immunoprecipitation analysis reveals that the origin-bound Cdc6 is stable throughout the meiotic cycle. Preliminary evidence suggests that this protection reflects a change in chromatin structure that occurs in meiosis. Using the cdc28-degron allele, we show that depletion of Cdc28 leads to stabilization of Cdc6 in the mitotic cycle, but not in the meiotic cycle. We show physical association between Cdc6 and the meiosis-specific hCDK2 homolog Ime2. These results suggest that under meiotic conditions, Ime2, rather than Cdc28, regulates the stability of Cdc6. Chromatin-immunoprecipitation analysis reveals that similarly to the mitotic cell cycle, Mcm2 binds origins in G1 and meiotic S phases, and at the end of the second meiotic division, it is gradually removed from chromatin.

Figure 1.

Cdc6 is required for premeiotic DNA replication. MATa/MATα cdc6-1/cdc6-1 (strain Y208, top two panels) and its isogenic cdc6–1/CDC6 (strain Y1466, bottom panel) cells were grown at 25°C in PSP2 to a titer of 1 × 10⁷ cells/ml. Cells were washed in water, resuspended in warm SPM, and incubated at either 25 or 35°C (time 0) (top). Nocodazole was added to the PSP2 culture to a final concentration of 10 μg/ml. After 2-h incubation, cells were washed and resuspended in SPM as described above (time 0). Samples were taken at 2 h (dashed gray line) and 48 h (solid black line) to process for FACS analysis.

Figure 2.

In wild-type cells, Cdc6 is degraded upon entry into premeiotic DNA replication. Wild-type diploid cells (strain Y1384) grown at 25°C in PSP2 to a titer of 1 × 10⁷ were washed in water, resuspended in SPM, and incubated at 25°C. Samples were taken at the indicated times for Western analysis (A), IP-Western (B), and FACS analysis (C). Early and late meiotic times are from two experiments. For comparison, the 9- and 10-h sample were taken from both experiments. The results in B and C are from a single experiment. Cdc6–13 × myc was detected using antibodies directed against the myc epitope. Cdc28 was detected using antibodies directed against the PSTAIRE epitope. C, control, without tagged protein. Sample taken from strain Y422 grown in PSP2 to 1 × 10⁷ cells/ml. Arrows in C indicate the position of cells with 2C and 4C DNA content.

Figure 5.

Origin-bound Cdc6 is stable throughout the meiotic cycle. Wild-type diploid cells (strain Y1384) grown at 25°C in PSP2 to a titer of 1 × 10⁷ were washed in water, resuspended in SPM, and incubated at 25°C. Samples were taken at the indicated times for immunoprecipitation (Figure 2B), FACS (Figure 2C), and ChIP analysis (this figure). For comparison, the 9- and 10-h sample were taken from both experiments. c, the strain without the myc tagged Cdc6 was Y422. WCE, whole cell extract before IP. The primers used to amplify the indicated DNA are described in MATERIALS AND METHODS.

Figure 4.

Cdc6 is degraded upon entry into premeiotic S phase. The percentage of cells with 4C DNA content was calculated from Figures 2 C and 3B (squares, filled line). The relative level of Cdc6 was calculated from Figures 2B and 3A (triangles, dashed line). (A) Wild-type strain. (B) ime2Δ strain.

Figure 3.

In IME2-deleted cells, Cdc6 is degraded upon entry into premeiotic DNA replication. ime2Δ/ime2Δ diploid cells (strain Y1385) grown at 25°C in PSP2 to a titer of 1 × 10⁷ were washed in water, resuspended in SPM, and incubated at 25°C. Samples were taken at the indicated times for Western (A) and FACS analysis (B). Early and late meiotic times are from two experiments. For comparison, the 10-h sample was taken from both experiments. Cdc6–13 × myc, Cdc28, and Mcm2 were detected using antibodies directed against the myc epitope, the PSTAIRE epitope, and Mcm2 N-terminus peptide, respectively. *, control, without tagged protein. Sample taken from strain Y1073 grown in PSP2 to 1 × 10⁷ cells/ml. Arrows in B indicate the position of cells with 2C, 4C, and >4C DNA content.

Figure 6.

Ectopic, elevated activation of Cdc28 results in premature entry into premeiotic S phase without affecting the stability of the origin-bound Cdc6. Wild-type (Y1384; A and B) and isogenic ime2Δ/ime2Δ (Y1385; C and D) diploid cells carrying pIME1-CLB1 ona2μ vector (YEp2053) grown at 25°C in PSP2 to a titer of 1 × 10⁷ were washed in water, resuspended in SPM, and incubated at 25°C. Samples were taken at the indicated times for FACS (A and C) and ChIP analysis (B and D). c, the strain without the myc tagged Cdc6 was Y422 (B) or Y1073 (D). WCE, whole cell extract before IP. Primers used to amplify origin (ARS305) and the nonorigin DNA are described in MATERIALS AND METHODS.

Figure 7.

Mcm2 is excluded from DNA origins during meiotic nuclear divisions. Diploid cells (strains Y1384) grown at 30°C in PSP2 to a titer of 1 × 10⁷ were washed in water, resuspended in SPM, and incubated at 30°C. Samples were taken at the indicated times for ChIP (A) and FACS analysis and DAPI staining (B). (A) Antibodies directed against Mcm2 were used. C, control without formaldehyde cross-linking. WCE, whole cell extract before IP. The PCR amplification was done using 1 μl of DNA from each sample. Top right panel shows that this quantity gives rise to a linear range PCR. (B) Percentage of cells with 4C DNA content calculated from the FACS analysis is illustrated (triangles). Percentage of binucleated (closed squares) and tetranucleated (open squares, dashed line) cells was determined after DAPI staining.

Figure 8.

Cdc28 is not required for the degradation of Cdc6 in the meiotic cycle. Diploid cells homozygous for cdc28-degron (strain Y1443) grown at 25°C in PSP2 to a titer of 1 × 10⁷ cells/ml were shifted to 37°C for 2 h (time 0). (A) G1-arrested cells were then shifted to either 25 or 35°C in PSP2 medium, for vegetative growth conditions. (B) G1-arrested cells were then washed, resuspended in SPM, and incubated at either 25 or 35°C for meiotic conditions. At the indicated hours, samples were taken to process for IP-Western by using anti-myc antibodies.

Figure 9.

Cdc6 and Ime2 physically associate. (A) Two-hybrid interaction. Level of β-galactosidase (given in Miller units) in a haploid strain (Y153) carrying pADH1-gal4(1-147)-HA-ime2K97A (YEp2225) with either pADH1-gal4(768-881)-HA (pGAD2F) or pADH1-gal4(768-881)-HA-CDC6 (YEp1349). Proteins were extracted from 1 × 10⁷ cells/ml grown in SD medium or from cells grown in SD medium to stationary, washed in water, and incubated in SPM medium at a titer of 1 × 10⁷ cells/ml for 6 h. (B) Coimmunoprecipitation. Proteins were extracted from cells incubated for 3 h in SPM. Anti-myc (lanes 1–4) or anti-Gal4(dbd) (lanes 5–8) immune complexes were prepared from the following strains: wild-type Y422 (lanes 1 and 2) or Y1384 (lanes 3 and 4) and ime2Δ (Y1385, lanes 5–8), diploid cells, carrying pIME1-gal4(dbd)-HA-ime2K97A (YEp2229) (lanes 2, 4, 6, and 8). Strains Y1384 and Y1385 (lanes 3–8) also carried CDC6–13 × myc. Proteins were separated on SDS-PAGE, and immunoblotting was done with anti-Gal4(dbd) or anti-myc, as indicated.