mcl1+, the Schizosaccharomyces pombe homologue of CTF4, is important for chromosome replication, cohesion, and segregation

Abstract

The fission yeast minichromosome loss mutant mcl1-1 was identified in a screen for mutants defective in chromosome segregation. Missegregation of the chromosomes in mcl1-1 mutant cells results from decreased centromeric cohesion between sister chromatids. mcl1+ encodes a beta-transducin-like protein with similarity to a family of eukaryotic proteins that includes Ctf4p from Saccharomyces cerevisiae, sepB from Aspergillus nidulans, and AND-1 from humans. The previously identified fungal members of this protein family also have chromosome segregation defects, but they primarily affect DNA metabolism. Chromosomes from mcl1-1 cells were heterogeneous in size or structure on pulsed-field electrophoresis gels and had elongated heterogeneous telomeres. mcl1-1 was lethal in combination with the DNA checkpoint mutations rad3delta and rad26delta, demonstrating that loss of Mcl1p function leads to DNA damage. mcl1-1 showed an acute sensitivity to DNA damage that affects S-phase progression. It interacts genetically with replication components and causes an S-phase delay when overexpressed. We propose that Mcl1p, like Ctf4p, has a role in regulating DNA replication complexes.

FIG. 1.

mcl1-1 shows defects in chromosome segregation and cohesion. (A and B) Mitotic phenotypes are identified in log-phase cultures of strains SPB32590 (wild type [wt]) and 545 (mcl1-1) fixed at permissive temperature (25°C) or of strain 545 grown for 8 h at the restrictive temperature (36°C). Panel A is a gallery of representative images for the five categories of mitotic phenotypes presented graphically in panel B. ch, chromatin. (C) mcl1-1 cells expressing α-tubulin-GFP were grown on YES agar pads. The top row of images shows GFP fluorescence, and the bottom row shows chromatin stained with Hoecsht 33342. Images represent an elapsed time of 30 min. Bar, ∼3 μm. (D) Twelve tetrads from mcl1-1^ts (strain 546) cross with bub1Δ::ura4⁺ (strain 549), photographed after 4 days of growth on YES agar at 25°C. Genotypes were determined by replica plating and are given as follows: wt, mcl1⁺ bub1⁺; b, bub1Δ::ura4 mcl1⁺; m, mcl1-1^ts bub1⁺; bm, bub1Δ::ura4 mcl1-1^ts). (E, F, and G) Differential interference contrast (top panels) and deconvolution microscopy with fluorescence optics (lower panels) were used to image living mcl1-1 (strain 594) and mcl1⁺ (strain 494) cells with a marked centromere, centromere I. (E) Binucleate cells show 2:0 and 1:1 segregation. (F) Segregation patterns were categorized as follows: one GFP spot in only one nucleus of two (1:0), one GFP spot in both nuclei (1:1), two GFP spots in only one nucleus of two (2:0), no segregation (cut), or more than two GFP spots present (other). (G) Representative images from the sister-chromatid cohesion study are shown. (H) Serial dilutions of mcl1-1 (strain 546), mcl1⁺ (strain 100), mad2Δ (strain 547), and cut12-1 (strain 246) plated onto YES agar and YES agar plus 12.5 μg of TBZ/ml. Bars, 5 μm.

FIG. 2.

mcl1⁺ is a member of a eukaryotic protein family. (A) The domain structure of Mcl1p and sequence comparison results among a select set of sepB family members are shown. Identity and similarity percentages are for comparisons over the entire length of the protein by using the Smith-Waterman algorithm (60). HMG, high-mobility group. (B) Clustal W alignment of the CTF4- and sepB-like proteins reveals three domains of high similarity in these aligned peptide sequences (2). S.p., S. pombe; A.n., A. nidulans; S.c., S. cerevisiae; H.s., Homo sapiens.

FIG. 3.

mcl1⁺ is an essential gene. (A) Tetrads dissected from sporulated heterozygous diploids result in two colonies and two microcolonies (panel 1, with tetrads placed horizontally). Replica plating to medium selective for uracil prototrophy demonstrated that none of the visible colonies carried the gene replacement (panel 2). Null cells generated a microcolony on YES agar and are shown in the image below panel 2 (bar, ∼20 μm). (B) Null spores were selectively germinated in medium lacking uracil. Cells were stained with DAPI and Calcofluor. The three panels show representative fields of cells from the selectively germinated mcl1Δ culture at t = 24, 48, and 72 h, with histogram plots from FACS below each panel.

FIG. 4.

Mcl1-GFP is a constitutive nuclear protein that associates with chromatin during G₁ and S phase but not during mitosis. (A) Hoechst 33342-stained DNA forms a three-quarters circle with two protruding bars, which are the rDNA genes extending into the nucleolus. Mcl1-GFP expressed from its endogenous promoter fills the nuclear volume but appears excluded from one region. Merged images show that exclusion is coincident with the rDNA and nucleolus. (B) Live cell fluorescence microscopy of cells stained with Calcofluor (top panels) and GFP fluorescence (bottom panels). There was no apparent redistribution of Mcl1-GFP fluorescence through the cell cycle, and any deformation in the nuclear membrane was filled with GFP fluorescence (white arrowheads), indicating that much of the Mcl1-GFP is freely diffusible in the nucleus. (C) Log-phase cells showing MTs in blue, DNA in red, and Mcl1-GFP in green. Using cell morphology to assess the cell cycle stage, we quantified the fraction of cells in which GFP and DNA colocalized after the extraction of soluble protein (see Materials and Methods). The data are presented just below the panel of images (n = 251). The far right panel is an image of cells treated with DNase I and then similarly extracted. The bar represents 5 μm. (D) Chemical and mutant cell cycle arrests were used to confirm log-phase results for the stage-specific localization of Mcl1-GFP to chromatin. We tested the retention of Mcl1-GFP in cdc10-129 (strain 561), orp1-4 (strain 557), cdc22-M45 (strain 558), HU (strain 551), cdc21-M68 (strain 556), cdc17-K42 (strain 559), cdc25-22 (strain 560), and TBZ mutants (strain 551) under arrested conditions (3 h at 36°C for mutants and 3 h at 32°C for chemical arrests). The percentages of cells (n > 200 each) in which GFP signal colocalized with DAPI are presented graphically.

FIG. 5.

Overexpression of Mcl1p causes a G₁/S arrest. (A) Wild-type cells (strain 100) transformed with pREP3x mcl1-GFP grow slowly when expression is induced. (B) FACS analysis of cells induced for 12 h show a modest G₁ accumulation in cells expressing high levels of mcl1-GFP compared with cells repressed for this expression. (C) Expression of Mcl1-PK in the hsk1-1312 (FY594) mutant under inducing conditions severely retarded growth of the mutant in comparison to hsk1-1312 mutant carrying an empty vector. However, this expression does not lead to increased cell death. Cells were grown for 4 days at 29°C. (D) hsk1-1312 cells under inducing conditions for 24 h have a shift to 1N DNA content when expressing mcl1-PK but not in the empty vector controls (C). Expression from the nmt promoter starts at time zero and reaches a maximum after 12 h (23). Data are representative of three independent transformations of hsk1-1312 mutants with pREP41x mcl1-PK1 and pREP41x mcl1-GFP.

FIG. 6.

mcl1-1 cells have an altered chromosome structure. (A) A total of 10⁶ cells (2 μg of DNA) per lane were prepared for PFGE analysis from mcl1⁺ and mcl1-1 cultures. mcl1⁺ cells were incubated at 25°C (1) and arrested with 12 mM HU for 3 h (2). mcl1-1 cells were incubated at 25°C (3), and mcl1-1 cells were shifted to incubation at 36°C for 4 (4), 8 (5), and 12 h (6). mcl1-1 cells were arrested for 3 h in 12 mM HU (7). cdc24-M38 cells were shifted to 36°C for 3 h of incubation (8) and by the S. pombe chromosome standards (9). (B) FACS profiles of mcl1-1 held at 36°C for 0, 4, 8, 12, and 24 h are shown. (C) A Southern blot of ApaI-digested genomic DNA isolated from tetrad-matched mcl1⁺ and mcl1-1 strains and grown to 100 generations at 25°C is shown. The blot was probed with a 300-bp subtelomeric fragment that detects the 3′ terminus of the telomere (4).

FIG. 7.

mcl1-1 requires the DNA damage checkpoint but is partially rescued by cds1Δ. (A) mcl1-1, cdc25-22, wee1-50, mcl1-1 cdc25-22, mcl1-1 wee1-50, and mcl1-1 bub1Δ cells were grown at 25°C to early log phase and shifted to the restrictive temperature of 36°C. Samples were taken every hour to determine viability upon return to the permissive temperatures. The graph presents means ± standard errors of the means from three independent experiments. (B) cds1Δ mcl1-1 double mutants and cds1Δ mcl1-1 chk1Δ triple mutants were constructed. Survival of mcl1-1 cells was enhanced under permissive conditions by the deletion of cds1⁺. (C) Cds1p kinase activity was determined by its ability to phosphorylate the amino terminus of Wee1p. Lanes: 1, log-phase mcl1⁺ pREP42xcds1⁺ (3× HA); 2, mcl1⁺ pREP42xcds1⁺ (3× HA) arrested with 12 mM HU for 3 h; 3, mcl1⁺ pREP42cds1-KD (3× HA) arrested with 12 mM HU for 3 h; 3, mcl1-1 pREP42xcds1⁺ (3× HA) in log phase; 4, mcl1-1 pREP42xcds1⁺ (3× HA) arrested with 12 mM HU for 3 h; 6, mcl1-1 pREP42xcds1KD (3× HA) arrested with 12 mM HU for 3 h. The top panel is the kinase assay, and the bottom panel is a Western blot with an anti-HA. IgG, immunoglobulin G. (D) Overexpression of either Cds1p or Cds1KDp from an inducible promoter was toxic to mcl1-1 cells. The mcl1⁺ (strain 99) and mcl1-1 (strain 546) isogenic strains carried pREP42xcds1⁺ (3× HA) and pREP42xcds1-KD (3× HA) grown on selective medium. Plasmid expression was repressed by the addition of thiamine and induced by its absence. Wt, wild type.

FIG. 8.

mcl1-1 is sensitive to DNA damage. (A) Wild-type (wt; strain 99), mcl1-1 (strain 546), cds1Δ (strain FY865), mcl1-1 cds1Δ (strain 563), and rad3Δ (strain FY1105) cells were exposed to the indicated γ-ray doses. Relative viability is plotted. (B) mcl1-1 cell growth is hypersensitive to the continual presence of 3 mM HU. The mcl1-1 cds1Δ mutant (strain 563) is more sensitive to HU than either single mutant, and its sensitivity is similar to that of the rad3Δ mutant (strain FY1105). (C) Only a checkpoint-defective strain, rad3Δ, failed to arrest cell division in response to 12 mM HU, as indicated by the septation index. (D) mcl1-1 cells are sensitive to a prolonged arrest in HU but not as sensitive as checkpoint-defective strains. (E) mcl1-1 cells are as sensitive to MMS as are rad3Δ cells and are unable to grow in the presence of 0.0025% MMS. Sensitivity of mcl1-1 cells is rescued by pREP42x expression of mcl1-GFP. (F) FACS analysis of the mcl1⁺(strain 99), rad3Δ (strain FY1105), and mcl1-1 (strain 546) cultures treated with 0.02% MMS is illustrated. mcl1-1 cells had a much weaker arrest in S phase than did mcl1⁺ cells (strain 99). This is similar to the rad3Δ phenotype. 1N and 2N reference peaks were generated by a mix of cdc10-129 and cdc25-22 cells arrested for 2.5 h at 36°C.