Fission yeast Eso1p is required for establishing sister chromatid cohesion during S phase

Abstract

Sister chromatid cohesion is essential for cell viability. We have isolated a novel temperature-sensitive lethal mutant named eso1-H17 that displays spindle assembly checkpoint-dependent mitotic delay and abnormal chromosome segregation. At the permissive temperature, the eso1-H17 mutant shows mild sensitivity to UV irradiation and DNA-damaging chemicals. At the nonpermissive temperature, the mutant is arrested in M phase with a viability loss due to a failure to establish sister chromatid cohesion during S phase. The lethal M-phase arrest phenotype, however, is suppressed by inactivation of a spindle checkpoint. The eso1(+) gene is not essential for the onset and progression of DNA replication but has remarkable genetic interactions with those genes regulating the G(1)-S transition and DNA replication. The N-terminal two-thirds of Eso1p is highly homologous to DNA polymerase eta of budding yeast and humans, and the C-terminal one-third is homologous to budding yeast Eco1p (also called Ctf7p), which is required for the establishment of sister chromatid cohesion. Deletion analysis and determination of the mutation site reveal that the function of the Eco1p/Ctf7p-homologous domain is necessary and sufficient for sister chromatid cohesion. On the other hand, deletion of the DNA polymerase eta domain in Eso1p increases sensitivity to UV irradiation. These results indicate that Eso1p plays a dual role during DNA replication. The C-terminal region acts to establish sister chromatid cohesion, and the N-terminal region presumably catalyzes translesion DNA synthesis when template DNA contains lesions that block regular DNA replication.

FIG. 1

Phenotypes of the eso1-H17 mutant. (A) The eso1-H17 mutant shows an elongated cell morphology. Wild-type (h⁻ leu1-32) and eso1-H17 (h⁻ eso1-H17 leu1-32) cells were inoculated on YEA plates, incubated overnight at the indicated temperatures, and observed under the microscope. (B) The eso1-H17 mutant is arrested with a broad peak of G₂ DNA content. Cells were grown to mid-log phase at 23°C in MM medium and shifted up to 34°C. Cells were sampled at 2, 4, 6, and 8 h after the temperature shift and analyzed by flow cytometry.

FIG. 2

Loss of Eso1p function leads to spindle assembly checkpoint-dependent mitotic delay. (A) eso1-H17 cells quickly lost viability upon a shift to the nonpermissive temperature. Wild-type (h⁻ leu1-32), eso1-H17 (h⁻ eso1-H17 leu1-32), Δmad2 (h⁻ mad2::ura4⁺ ura4-D18 leu1-32), and eso1-H17 Δmad2 (h⁻ eso1-H17 mad2::ura4⁺ ura4-D18 leu1-32) cells were grown to mid-log phase at 25°C in YEL medium and shifted to 36°C. Cell aliquots were taken at 4 and 8 h after the temperature shift and plated on YEA at 25°C. (B) eso1-H17 cells are defective in chromosome segregation. Cells were grown to mid-log phase at 25°C in YEL medium and shifted to 36°C. Cell aliquots were taken at indicated times, fixed with glutaraldehyde, and stained with DAPI. Arrowheads indicate the cells showing a mitotic delay. Bar, 10 μm. (C) Temperature sensitivity of eso1-H17 cells is partially suppressed by deletion of the mad2⁺ gene. The indicated cells were inoculated on YEA plates and incubated for 3 days at the indicated temperatures.

FIG. 3

eso1-H17 cells are sensitive to DNA DSB. (A) eso1-H17 cells are sensitive to UV irradiation. Wild-type (h⁻ leu1-32) and eso1-H17 (h⁻ eso1-H17 leu1-32) cells were plated on YEA, irradiated with various doses of UV, and incubated for 7 days at 27°C. (B) eso1-H17 cells are sensitive to MMS and bleomycin but not to 4NQO. Approximately 10⁴, 10³, 10², and 10 cells of the wild type (upper spots) and the eso1-H17 mutant (lower spots) were spotted on normal YEA plates (referred to as control) or YEA plates containing MMS (0.004% [vol/vol]), bleomycin (0.025% [wt/vol]), or 4NQO (0.04 μM). Plates were incubated at 27°C for 3 days (control) or 4 days (others).

FIG. 4

Eso1p is required for the establishment of sister chromatid cohesion during S phase but not for its maintenance in G₂ and M phases. (A) DNA replication takes place without any significant delay in eso1-H17 mutant cells. Wild-type (h⁻ leu1-32) and eso1-H17 (h⁻ eso1-H17 leu1-32) cells were grown to mid-log phase at 25°C and then nitrogen starved in MM-N medium for 24 h to be arrested in G₁. Cells were then released by transfer into MM medium preincubated at 36°C. Cell aliquots were taken every hour and analyzed for S-phase onset and progression by flow cytometry. (B) eso1-H17 cells lose viability during S phase. To determine cell number (open squares), eso1-H17 cell aliquots were taken every hour and counted. To determine cell viability (open circles), eso1-H17 cell aliquots taken every hour were plated on YEA at 25°C. To determine percent abnormal nuclear cells (filled triangles), cells were fixed with 70% ethanol and stained with DAPI, and those with abnormal chromosome structures (overcondensed chromosomes, cut, missegregation, etc.) were counted under the fluorescence microscope. (C and D) Sister chromosomes are prematurely separated in eso1-H17 mutant cells. Wild-type (h⁺ Cen1-GFP) and eso1-H17 (h⁺ eso1-H17 Cen1-GFP) cells were arrested in G₁ by nitrogen starvation at 26°C and then released in MM medium preincubated at 36°C. Live cells were observed under the fluorescence microscope. (C) Frequencies of cells showing two split Cen1-GFP signals. (D) Examples of eso1-H17 cells showing two split Cen1-GFP signals and wild-type control at 4 h. Bar, 5 μm. (E and F) Eso1p is not essential for the maintenance of cohesion in G₂ and M phases. Cells of eso1-H17 (h⁻ eso1-H17 leu1-32) and rad21-K1 (h⁻ ura4-D18 rad21-K1-ura4⁺ leu1-32) mutants were grown to saturation in YEL medium at 25°C and incubated for a further 24 h to ensure growth arrest. They were then incubated at 36°C for 3 h and released in fresh YEL medium preincubated at 36°C. (E) Flow cytometric analysis of arrested cells. (F) Lack of abnormal mitosis in eso1-H17 cells released from G₂ phase at the nonpermissive temperature. Cell number (open squares) was determined by counting aliquots taken every 30 min. To determine percent abnormal mitosis (filled triangles), cells were fixed with 70% ethanol and stained with DAPI, and those with abnormal mitotic chromosome structures (cut, missegregation, etc.) were counted under the fluorescence microscope.

FIG. 5

Isolation and characterization of the eso1⁺ gene. (A) Restriction map of the eso1⁺ gene. The eso1⁺ open reading frame is shown by an arrow, and the Eco1p/Ctf7p-homologous domain is shaded. The EcoRI-PstI region of eso1⁺ was replaced with a ura4⁺ gene cassette for generating an eso1 null mutant. (B) Schematic illustration of Eso1p of S. pombe and Rad30p and Eco1p/Ctf7p of S. cerevisiae. The zinc finger motifs are filled. (C) Amino acid homologies between Eso1p, Rad30p, and Eco1p/Ctf7p. The predicted amino acid sequence of Eso1p is shown in a single-letter code and aligned with Rad30p and Eco1p/Ctf7p. Identical amino acids are boxed. The zinc finger motifs are underlined. (D) Cells with deletions of eso1⁺ are lethal. Spores from eso1⁺/eso1::ura4⁺ diploid cells were tetrad dissected on YEA plates and incubated at 30°C for 4 days. (E) Terminal phenotype of Δeso1 cells. Cells that germinated from two independent Δeso1 spores on a YEA plate were photographed. (F) DAPI staining of germinating Δeso1 cells. Δeso1 spores (Ura⁺) derived from eso1⁺/eso1::ura4⁺ diploid cells were preferentially germinated in MM lacking uracil. Germinating cells were fixed with 70% ethanol, stained with DAPI, and photographed under the microscope.

FIG. 5

Isolation and characterization of the eso1⁺ gene. (A) Restriction map of the eso1⁺ gene. The eso1⁺ open reading frame is shown by an arrow, and the Eco1p/Ctf7p-homologous domain is shaded. The EcoRI-PstI region of eso1⁺ was replaced with a ura4⁺ gene cassette for generating an eso1 null mutant. (B) Schematic illustration of Eso1p of S. pombe and Rad30p and Eco1p/Ctf7p of S. cerevisiae. The zinc finger motifs are filled. (C) Amino acid homologies between Eso1p, Rad30p, and Eco1p/Ctf7p. The predicted amino acid sequence of Eso1p is shown in a single-letter code and aligned with Rad30p and Eco1p/Ctf7p. Identical amino acids are boxed. The zinc finger motifs are underlined. (D) Cells with deletions of eso1⁺ are lethal. Spores from eso1⁺/eso1::ura4⁺ diploid cells were tetrad dissected on YEA plates and incubated at 30°C for 4 days. (E) Terminal phenotype of Δeso1 cells. Cells that germinated from two independent Δeso1 spores on a YEA plate were photographed. (F) DAPI staining of germinating Δeso1 cells. Δeso1 spores (Ura⁺) derived from eso1⁺/eso1::ura4⁺ diploid cells were preferentially germinated in MM lacking uracil. Germinating cells were fixed with 70% ethanol, stained with DAPI, and photographed under the microscope.

FIG. 6

The carboxyl-terminal Eco1p/Ctf7p-homologous domain of Eso1p is essential to rescue the eso1-H17 mutant. N-terminally and C-terminally truncated eso1⁺ genes were constructed and assayed for the ability to rescue the eso1-H17 mutant. The intact Eso1p is shown at the top (referred to as full). The Eco1p/Ctf7p-homologous domain is grey, and the zinc finger motifs are black. The amino acid numbers are shown at each mutant. The ability of the deletion mutants to complement eso1-H17 cells is shown in the right columns. For complementation activity, see Materials and Methods.

FIG. 7

Phenotypes of eso1 null mutants suppressed by amino-terminally truncated eso1⁺. (A) Nuclear morphologies of Δeso1 cells (h⁻ ade6-M210 eso1::ura4⁺ ura4-D18 leu1-32) suppressed by full-length eso1⁺ (pcL-full) or its amino-terminal truncation mutants (pcL-ΔN458 and pcL-ΔN597). Cells were grown to mid-log phase at 30°C in YEL medium, fixed with glutaraldehyde, and stained with DAPI. (B) Bleomycin and UV sensitivities. Approximately 5 × 10⁴, 5 × 10³, 5 × 10², and 50 cells of the wild type (h⁻ ade6-M210) and the Δeso1 mutant (h⁻ ade6-M210 eso1::ura4⁺ ura4-D18 leu1-32) suppressed by pcL-full, pcL-ΔN458, and pcL-ΔN597 were spotted on normal YEA plates (referred to as control), YEA plates containing bleomycin (0.02% [wt/vol]), or normal YEA plates but with irradiation with UV (100 J/m²). Plates were incubated at 30°C for 3 days. (C) Growth curves. Δeso1 cells (h⁻ ade6-M210 eso1::ura4⁺ ura4-D18 leu1-32) suppressed by pcL-full, pcL-ΔN458, or pcL-ΔN597 were grown at 30°C in MM plus adenine. (D) UV sensitivity. Δeso1 cells (h⁻ ade6-M210 eso1::ura4⁺ ura4-D18 leu1-32) suppressed by pcL-full, pcL-ΔN458, or pcL-ΔN597 were plated on YEA, irradiated with various doses of UV, and incubated for 5 days at 30°C.

FIG. 8

eso1⁺ genetically interacts with the genes encoding adherin and cohesin subunits. (A) Wild-type (h⁻ leu1-32), eso1-H17 (h⁻ eso1-H17 leu1-32), mis4-242 (h⁻ mis4-242 leu1-32), and eso1-H17 mis4-242 (h⁻ eso1-H17 mis4-242 leu1-32) cells were incubated on YEA plates for 3 to 4 days at the indicated temperatures. (B) rad21-K1 (h⁺ ade6-M216 ura4-D18 rad21-K1-ura4⁺ leu1-32) and eso1-H17 rad21-K1 (h⁻ ade6-M216 ura4-D18 eso1-H17 rad21-K1-ura4⁺ leu1-32) cells that were transfected with pREP81-rad21⁺ were inoculated on an MM-adenine plate (− thiamine) and an MM-adenine plate containing 10 μM thiamine (+ thiamine) and incubated for 6 days at 23°C.