Genetic interaction involving Swi1-Swi3, RFC^Ctf18, and Chl1. (A) None of the viable spores from swi1::Kan^r x ctf18::his3⁺, chl1::Kan^r × ctf18::his3⁺, and swi3::Kan^r × ctf18::his3⁺ crosses were able to grow on YES medium containing G-418 and EMM2, Edinburgh minimal media lacking histidine, indicating that swi1Δ ctf18Δ, chl1Δ ctf18Δ, and swi3Δ ctf18Δ cells are inviable. Genotypes of viable spores are shown. Representative images of >40 tetrad dissections from each cross are shown. (B) Damage sensitivities of swi1Δ were suppressed by an increased gene dosage of chl1⁺ or ctf18⁺. swi1Δ or ctf18Δ cells were transformed with the indicated plasmid and plated on YES medium containing no drug (YES), 5 mM HU, or 0.005% MMS. Fivefold serial dilution of cells were plated and incubated for 2–3 d at 32°C. Representative images of repeat experiments are shown. (C) Summary of genetic interaction involving Swi1-Swi3, RFC^Ctf18, and Chl1. (D) Ctf18 associates with Rfc4 and Rfc5. Protein extracts were prepared from cells expressing the indicated fusion proteins. Ctf18-TAP was precipitated and probed with anti-myc antibodies. Rfc4-Myc and Rfc5-Myc are shown to be equally expressed in each cell line (whole-cell extract, WCE). As a positive control, Rad17-TAP is shown to associate with Rfc4 and Rfc5. IgG IP, precipitated fraction.

Ctf18 constitutes a checkpoint-independent S phase DNA damage response pathway. (A and B) Fivefold serial dilution of cells were incubated on YES agar medium supplemented with the indicated amounts of MMS or CPT for 2–4 d at 32°C. Representative images of repeat experiments are shown.

Ctf18 associates with chromatin during S phase. (A) Diagram of the ori2004 region on S. pombe chromosome II used in ChIP is shown. (B) ChIP assay of Ctf18-5FLAG were performed at ori2004, at sites located 14 or 30 kb away from this origin and at ori3002 as indicated. cdc25-22 cells were synchronized at the G2-M boundary by incubation at 36°C for 4 h and then released into fresh YES medium containing 0 or 0.03% MMS at 25°C as indicated. ChIP assays were performed at the indicated times. ChIP of input (whole-cell extract) samples shows that 3 PCR products at ori2004 amplify equally with the primers. Representative results from repeat experiments are shown. (C) An increase in the septation index indicates the onset of S phase.

Ctf18, Chl1, and FPC play an important role for proper sister chromatid cohesion. (A) Cells of the indicated genotypes that had LacO repeats near centromere 1 and expressed LacI-GFP-NLS were grown to mid-log phase and supplemented with 10 mM HU for 2 h to synchronize cells in early S phase. Cells were then released into fresh YES medium containing 100 μg/ml TBZ for 3, 4, and 5 h to obtain metaphase cells. Representative images at 4 h in TBZ are shown for cells of the indicated genotypes. (B) Quantification of metaphase cells that had two GFP foci shown in A. At least 200 cells were counted for each strain. Error bar corresponds to the standard deviations obtained from three independent experiments. (C) cdc25-22 cells with the indicated genotypes were arrested at G2-M and released into the cell cycle in the presence of 100 μg/ml TBZ. Quantification of metaphase cells that had two GFP foci was performed at the indicated times as described above. (D) nda3-KM311 cells with the indicated genotypes were arrested at prophase, and cells with two GFP foci was quantified at the indicated times as described above. (E) Swi1-Swi3 and RFC^Ctf18 are required for cellular tolerance to a microtubule drug, TBZ. Fivefold serial dilution of cells of the indicated genotypes were incubated on YES agar medium supplemented with 0 or 20 μg/ml TBZ for 3 d at 30°C. (F) Genetic interaction between swi1, chl1 and ctf18. swi1Δ or ctf18Δ cells were transformed with the indicated plasmid and plated on YES medium containing 0 or 15 μg/ml TBZ. Fivefold serial dilution of cells were incubated for 2–3 d at 32°C. Representative images of repeat experiments are shown.

Ctf18 is involved in the Cds1-dependent replication checkpoint. (A and B) Synergistic interaction of ctf18Δ and chk1Δ in UV and HU survival assays indicates that Ctf18 is required for cellular tolerance to fork arrest. For UV survival assays, fivefold serial dilution of cells were plated on YES agar medium and exposed to the indicated doses of UV. Agar plates were then incubated for 2–3 d at 32°C. For HU sensitivity assays, fivefold serial dilution of cells were incubated on YES agar medium supplemented with the indicated amounts of HU for 2–4 d at 32°C. Representative images of repeat experiments are shown. (C) Cds1 activation is strongly reduced in ctf18Δ cells. Cells of the indicated genotypes were incubated in YES liquid medium supplemented with 12 mM HU for 0, 1, 2, and 4 h at 30°C. Kinase activity of immunoprecipitated Cds1 was measured using MBP as a substrate. The radiolabeled MBP was detected after gel electrophoresis (top). The radioactivity levels (cpm) of MBP were then determined in a liquid scintillation counter (bottom). Representative results from repeat experiments are shown.

Ctf18 is involved in stabilization of replication forks. (A) Rad22-YFP foci formation was significantly elevated in ctf18Δ cells. Cells of the indicated genotype expressing genomic Rad22-YFP were grown in YES medium at 25°C until mid-log phase. The percentages of nuclei with at least one Rad22-YFP focus are shown. The standard deviations were obtained from four independent experiments (B) Quantification of Rad22-YFP foci according to cell cycle stages. S and early G2 cells had the most Rad22-YFP foci. The percentages of nuclei that have at lest one focus or harbor two or more foci are shown. At least 200 cells were counted for each strain. Error bar corresponds to the standard deviation obtained from four independent experiments. (C) Schematic drawing for nuclear and morphological changes during the S. pombe cell cycle. (D) Ctf18 is required for the efficient resumption of replication after fork arrest. Chromosome samples from either wild-type or ctf18Δ cells were examined by PFGE. Cells were grown until mid-log phase and then incubated in the presence of 12 mM HU for 3 h at 30°C. Cells were then washed and released into fresh medium. Chromosomal DNA samples were prepared at the indicated times. Representative results from repeat experiments are shown.

Chl1 is involved in an S phase response pathway. (A and B) For drug sensitivity assays, fivefold serial dilution of cells were incubated on YES agar medium supplemented with the indicated amounts of MMS, CPT, HU, or TBZ for 2–4 d at 32°C. For UV survival assays, fivefold serial dilution of cells of the indicated genotypes were spotted onto YES agar medium and exposed to the indicated doses of UV irradiation. The plates were then incubated for 2–3 d at 32°C. Representative images of repeat experiments are shown.

Models for S phase stress response mechanisms in S. pombe. RFC^Ctf18 is involved in Cds1-dependent replication checkpoint. FPC^Swi1-Swi3, RFC^Ctf18, and Chl1 also have checkpoint independent functions that are important for fork protection and DNA repair. In this model, FPC^Swi1-Swi3, RFC^Ctf18, and Chl1 stabilize replication forks in a configuration that is recognized by replication checkpoint sensors. Chl1 may work together with FPC^Swi1-Swi3to facilitate fork protection. In addition, FPC^Swi1-Swi3and RFC^Ctf18 act in parallel to facilitate proper chromosome cohesion.