Cdc18/CDC6 activates the Rad3-dependent checkpoint in the fission yeast

Abstract

A screen for genes that can ectopically activate a Rad3-dependent checkpoint block over mitosis in fission yeast has identified the DNA replication initiation factor cdc18 (known as CDC6 in other organisms). Either a stabilized form of Cdc18, the Cdc18-T6A phosphorylation mutant, or overexpression of wild type Cdc18, activate the Rad3-dependent S-M checkpoint in the apparent absence of detectable replication structures and gross DNA damage. This cell cycle block relies on the Rad checkpoint pathway and requires Chk1 phosphorylation and activation. Unexpectedly, Cdc18-T6A induces changes in the mobility of Chromosome III, affecting the size of a restriction fragment containing rDNA repeats and producing aberrant nucleolar structures. Recombination events within the rDNA appear to contribute at least in part to the cell cycle delay. We propose that an elevated level of Cdc18 activates the Rad3-dependent checkpoint either directly or indirectly, and additionally causes expansion of the rDNA repeats on Chromosome III.

Figure 1.

Overexpression of Cdc18 induces a Rad3-dependent cell cycle arrest with no evidence of re-replication. (A) Schematic of the screen. (B) Rad3^ts cells containing pRep4X cdc18 were grown on selective media plus thiamine (OFF) for 5 days at 25°C and then replica-plated onto selective media without thiamine (ON) at 25 and 36°C. Left panel: microscopy after 24 h at 25°C. Right panel: microscopy after 24 h at 36°C. (C) A culture of rad3^ts cells containing pRep4X cdc18 was grown for 24 h in selective media plus thiamine (OFF) before resuspension of cells in selective media minus thiamine for 11 h (ON). Half the culture remained at 25°C, the other half shifted to 36°C, for 8 h. Samples were taken every 2 h for microscopy and FACS analysis. Left panel: 25°C with no re-replication. Right panel: 36°C with no re-replication. (D) A re-replicating control strain (rad3+ pRep3X cdc18) was grown for 24 h at 32°C in selective media plus thiamine (OFF) before resuspension of cells in selective media minus thiamine for 11 h (ON). Samples were taken every 2 h for FACS analysis. Note the increasing DNA content with time.

Figure 2.

Stabilization of Cdc18 levels by mutation of the CDK consensus sites induces a Rad3-dependent cell cycle arrest in the absence of replication forks. (A) Top panel: a rad3^ts strain (CCL9) expressing the cdc18-T6A mutant from the endogenous locus was constructed and grown on plates in minimal medium at 36 or 25°C. Bottom panel: cells from a liquid culture of the same strain grown at 36°C and then shifted at 25°C for 6 h were fixed and stained with DAPI. (B) FACS analysis was performed on the same samples and corresponding control strains (CCL1, CCL3) as indicated. (C) Cdc25-22 cells were grown overnight at 25°C before shifting to 36.5°C to use as a control for length and FACS. Samples were taken every 2 h for the next 10 h for FACS analysis and DAPI staining. Elongated cells after 2 h in the G2 block are shown to demonstrate equivalent nuclear staining at an equivalent length to CCL9. Note the FACS after 2 h in the G2 block is also equivalent to that seen in the presence of the Cdc18-T6A mutant protein. (D) 2D-gel of genomic DNA extracted from cdc25-22 rad3Δura4 Cdc18-T6A (CCL13) was probed for ars3001. This strain behaves as wild-type cdc25⁺ at the permissive temperature of 25°C, but as cdc25⁻ at the restrictive temperature of 36.5°C. Samples were collected from cycling cells at 25°C (left) or from G2 arrested cells after incubation for 3 h at 36.5°C (right). Note absence of replicating structures in the G2 arrested culture.

Figure 3.

Different effects may relate to different levels of Cdc18 overexpression. Wild type (rad3⁺) cells, wild type (rad3⁺) cells containing pRep3X cdc18, and rad3^ts cells containing pRep4X cdc18, were cultured for 24 h in the presence (OFF) or in the absence (ON) of thiamine. Wild type (rad3⁺) and rad3^ts cdc18-T6A cells were also cultured for 24 h. Total protein lysates were separated on 10% SDS-PAGE and blotted on nitrocellulose membrane. The membrane was probed with polyclonal anti-Cdc18 antibodies (32). Note that the Cdc18-T6A band is higher in the gel than wild-type Cdc18 due to presence of the TAP tag. Note all lanes are from the same gel, separated to distinguish the slower running TAP-tagged Cdc18-T6A from wild Cdc18.

Figure 4.

Mutation of the CDK consensus of Cdc18 results in Rad3-dependent cell cycle arrest and Chk1 activation. (A) A rad3^ts cdc18-T6A (CCL9) strain was incubated for 4 h at 25°C to block cells at the G2-M transition. Cells were released by shifting the culture for 3 h to 36°C (schematic). From the time of the shift to 36°C, samples were taken every 20 min and cell cycle progression followed by FACS analysis (bottom right). The percentage of binucleated cells and the septation index were determined (bottom left). (B) A rad3^ts cdc18-T6A chk1-HA (CCL12) culture was synchronized and released as in A, but shifted back to 25°C after 1 h to re-impose the mitotic block. Total protein lysates, from samples taken every 20 min from release, were prepared and separated by SDS-PAGE followed by western blotting using anti-HA antibodies. (C) Strains CCL15-24 confirmed that the Cdc18-activated Rad3-dependent cell cycle arrest acts through the Chk1/Crb2 pathway, and not Cds1/Mrc1. Wild-type growth of the cdc18-T6A mutant was seen in the absence of all the Rad checkpoint proteins, and of Chk1 and Crb2. However, elongation was observed in the absence of Cds1 and Mrc1. Rad3Δ::ura4⁺ cdc18-T6A (CCL14), which grows normally at all temperatures, was used as a control.

Figure 5.

Mutation of the CDK consensus of Cdc18 does not affect S phase entry or duration. (A) A cdc25-22 rad3Δ::ura4⁺ cdc18-TAP (CCL8) and a cdc25-22 rad3Δ::ura4⁺ cdc18-T6A-TAP (CCL13) strains were synchronized in late G2 by a 4 h incubation at 36°C. After release to the permissive temperature of 25°C, samples were taken every 20 min and cell cycle progression was followed by flow cytometry analysis. (B) Samples were also taken every hour from the start of the G2 arrest at 36°C for measurement of cell number (left panel) and every 20 min from release for measurement of septation index. (C) Total protein lysates from samples from A were prepared and separated by SDS-PAGE followed by western blotting using the PAP antibody or anti-tubulin. AS = asynchronous population.

Figure 6.

The Cdc18 phosphorylation mutant affects the mobility of chromosome III the cell cycle. (A) Chromosome III not visualized in the presence of Cdc18-T6A on ethidium bromide staining, either in asynchronous cycling cells (CCL9) (left panel) or G2 arrested cells (CCL13) (right panel), but is present in the corresponding controls (CCL3 and CCL7). (B) Southern blotting and probing with non-origin rDNA (for a fragment downstream of ars3001) (top panel) and ade6 (bottom panel) demonstrate chromosome III is present in the gel as a smear. (C) Cdc25-22 rad3Δ::ura4⁺ cdc18-T6A cells (CCL13) were synchronized in G2 by growing overnight at 25°C and then shifting to 36.5°C for 3.5 h. Cells were then released by shifting back to 25°C and samples taken for septation index (top panel) and PFGE (middle panel) every 20 min for 3 h. The PFG was Southern blotted and probed with non-origin rDNA for chromosome III (bottom panel). CCL1 = wt.

Figure 7.

The changes in the size and variability of chromosome III disappear with removal of the Cdc18 phosphorylation mutant. (A) Ethidium bromide stained PFG of cross-derived strains not containing cdc18-T6A: 1–4 rad3⁺ cdc25⁺ isolates; 5–7 rad3Δ cdc25⁺ isolates; 8–10 rad3⁺ cdc25-22 isolates; 11,12 rad3Δ cdc25-22 isolates. (B) Southern blotting and probing of A for chromosome III with non-origin rDNA. (C) Southern blotting and probing for chromosome III with non-origin rDNA in four of the strains from A. Top panel: overnight culture before chromosomal agarose plug preparation. Bottom panel: 30 generation culture before chromosomal agarose plug preparation. CCL1 = wt.

Figure 8.

There is an expansion in the restriction fragments containing the rDNA repeats within chromosome III in the presence of Cdc18-T6A. (A) Agarose plugs containing chromosomes with wild-type cdc18 from the rad3^ts strain (CCL3), and with cdc18-T6A in the rad3^ts background (CCL9), underwent Sfi1 digest prior to PFGE under conditions to resolve fragments in kilobase (Kb) range. Southern blotting of and probing for chromosome III with non-origin rDNA followed. (B) As in A except PFGE was run under conditions to resolve fragments in megabase (Mb) range. CCL3 = wt.

Figure 9.

Cdc18-T6A induces inappropriate recombination in the rDNA repeats. (A) Gar2-GFP and Gar2-GFP Cdc18-T6A were observed in live cells using fluorescence microscopy. Arrows indicate abnormal nucleolar structures. Note: top panel Gar2-GFP only; bottom panel co-staining with Gar2-GFP and DAPI. (B) Left panel: strains with the indicated background (all containing the rad3^ts allele) were grown at 36°C and shifted to 25°C for 6 h. Right panel: the percentage of cells (200 observed per strain) elongated greater than twice wild-type length.