Fission yeast F-box protein Pof3 is required for genome integrity and telomere function

Abstract

The Skp1-Cullin-1/Cdc53-F-box protein (SCF) ubiquitin ligase plays an important role in various biological processes. In this enzyme complex, a variety of F-box proteins act as receptors that recruit substrates. We have identified a fission yeast gene encoding a novel F-box protein Pof3, which contains, in addition to the F-box, a tetratricopeptide repeat motif in its N terminus and a leucine-rich-repeat motif in the C terminus, two ubiquitous protein-protein interaction domains. Pof3 forms a complex with Skp1 and Pcu1 (fission yeast cullin-1), suggesting that Pof3 functions as an adaptor for specific substrates. In the absence of Pof3, cells exhibit a number of phenotypes reminiscent of genome integrity defects. These include G2 cell cycle delay, hypersensitivity to UV, appearance of lagging chromosomes, and a high rate of chromosome loss. pof3 deletion strains are viable because the DNA damage checkpoint is continuously activated in the mutant, and this leads to G2 cell cycle delay, thereby preventing the mutant from committing lethal mitosis. Pof3 localizes to the nucleus during the cell cycle. Molecular analysis reveals that in this mutant the telomere is substantially shortened and furthermore transcriptional silencing at the telomere is alleviated. The results highlight a role of the SCF(Pof3) ubiquitin ligase in genome integrity via maintaining chromatin structures.

Figure 1

Structure of the Pof3 F-box protein. (A) Overall structural organization. Schematic comparison of Pof3 and three other F-box proteins (fission yeast Pop1 and Pop2 and budding yeast Fcl1) is depicted. F in squares shows the F-box, whereas three other protein–protein interaction domains are indicated as follows: WD-repeats (ovals), TPR (squares), and LRRs (hexagons). (B and C) Alignment of amino acid sequence of the F-box (B) and TPR (C). Identical amino acids are shown in black squares with open letters, whereas conservative amino acids are shown in dark squares with black letters. Amino acid number and consensus amino acid residues are shown to the left and at the bottom, respectively. (D) Sequence alignment of LRRs. Consensus amino acid residues are boxed, and a repeating unit is shown below with consensus leucine and asparagine residues.

Figure 2

Complex formation between Pof3 and core components of the SCF ubiquitin ligase. (A) Interaction between Pof3 and cullin-1. Protein extracts were prepared from untagged wild-type (TP108-3A; Table 1, lanes 1 and 4), a singly tagged (SKP414-17, pcu1⁺- 13myc, lanes 2 and 5), or doubly tagged strain (SKP525, pof3⁺-3HA pcu1⁺-13myc, lanes 3 and 6), and immunoprecipitation was performed with anti-HA (lanes 1–3) or with anti-Myc antibody (lanes 4–6). After running on SDS-PAGE, immunoblotting was performed with anti-HA (top) or anti- Myc antibody (bottom). (B) Interaction between Pof3 and Skp1. Protein extracts were prepared from untagged wild-type (lanes 1 and 3) or a Pof3- 13Myc strain (SKP512, lanes 2 and 4), and immunoprecipitation was performed with anti-Skp1 antibody (lanes 3 and 4), followed by immunoblotting with anti-Myc antibody. Total extracts were also run (lanes 1 and 2).

Figure 3

Gene disruption of pof3⁺ results in cell elongation and cell cycle delay. (A) Tetrad analysis of diploid heterozygous for pof3. Three tetrads dissected from heterozygous diploid (SKDP521) are shown. In each tetrad, two normal-sized and two small- sized colonies are formed, of which small colonies are pof3 deleted. (B) Cell morphology of Δpof3 cells. Cells taken from one set of tetrads (A, 1a–d) were grown on rich media plates for 1 d. Bar, 10 μm.

Figure 4

DNA damage checkpoint is activated in the absence of Pof3. (A) Synthetic lethality between Δpof3 and ts rad3 mutants. Cells of wild-type (HM123), rad3^ts, Δpof3 (SKP471), or Δpof3rad3^ts (SKP459) were streaked on rich media plates and incubated at 36°C for 2 d. (B) Loss of viability in Δpof3rad3^ts. Exponentially growing cultures of four strains shown in A (wild type, squares; rad3^ts, diamonds; Δpof3, circles; Δpof3rad3^ts, triangles) were shifted from 26 to 36°C. Cell number was measured at each time point and viability was examined by plating cells on rich media plates after appropriate dilution. After incubating plates at 26°C, the number of colonies was counted and viability was calculated by dividing the number of viable colonies by the cell number. (C) Checkpoint defective phenotypes of Δpof3rad3^ts. Cells of Δpof3 (left) or Δpof3rad3^ts (right) grown at 26°C were shifted to 36°C, and incubated for 3 h, and nuclear DNA was stained with DAPI. Arrowheads show cut cells, whereas arrows emphasize cells with defects in chromosome segregation (see text; Figure 6, B and C). Bar, 10 μm.

Figure 5

DNA is damaged in pof3 disruptants. (A) Detection of thymine dimers. Wild-type and Δpof3 cells collected on filter papers were irradiated with UV (100 J/m²) and resuspended in fresh liquid medium at 26°C. Genomic DNA was prepared before and after UV irradiation (up until 4 h) and blotted onto a nitrocellulose filter (top) and also onto parafilm (bottom). The filter was immunoblotted with specific anti-thymine dimer antibody (TDM-2) (Mori et al., 1991), whereas DNA spotted on Parafilm (bottom) was stained with ethidium bromide (ETBr) to ensure equal loading of DNA. (B) Phosphorylation of Chk1. Wild-type (SKP467-13, lanes 2 and 3) and Δpof3 cells (SKP472, lanes 4 and 5) tagged with Chk1- 13Myc were grown exponentially and collected on two nitrocellulose filters. One filter of each strain was irradiated with UV (100 J/m²). After 20-min incubation in rich liquid medium for recovery, protein extracts were prepared from with (lanes 3 and 5) and without irradiation (lanes 2 and 4) and immunoblotting was performed with anti-Myc antibody. Immunoblotting was also performed in extracts from a nontagged wild-type strain (lane 1) as a negative control. Bands corresponding to phosphorylated (top) and nonphosphorylated Chk1 are marked. (C) Hypersensitivity to UV. The three strains indicated (wild-type, squares; Δrad3, diamonds; Δpof3, circles) were plated on rich medium and irradiated with various doses of UV. The number of viable colonies was counted after 4-d incubation at 26°C. Vertical axis (%) is shown logarithmically. (D) Normal responses to DNA damage in Δpof3. The three strains indicated were grown in rich liquid culture in the presence of phleomycin (10 μg/ml) at 26°C for 5 h, and cell morphology was observed. Bar, 10 μm. (E) Normal sensitivity to HU. Cells of wild-type, Δrad3 or Δpof3 were spotted onto rich media plates in the presence (right) or absence (left) of 2 mM HU, as serial dilutions (∼10⁵ cells in the left row in each lane and then diluted 10-fold in the subsequent rightward spots) and incubated at 30°C for 3 d.

Figure 6

High frequency of minichromosome loss and chromosome segregation defects in the absence of Pof3. (A) Loss of minichromosomes. Wild-type (CN2, top) and Δpof3 cells (SKP491, bottom) containing minichromosomes, which had been grown in minimal medium without adenine (selective conditions for minichromosomes), were streaked on rich media plates and incubated at 30°C for 4 d (left). Colonies of adenine auxotroph appeared as red colonies. Quantification of chromosome loss rate is shown on the right-hand side. (B) Chromosome segregation defects. Exponentially growing Δpof3 was stained with Hoechst 33342. Unequally segregated chromosomes are marked with arrows. Wild-type control is shown on the right-hand side. (C) Visualization of lagging chromosome. Live cells of a wild-type or Δpof3 strain containing integrated cut12⁺-GFP (green; Bridge et al., 1998) are stained with Hoechst 33342 (red) and viewed by fluorescence microscopy to observe chromosomes and the spindle pole body. Bar, 10 μm. (D) Hypersensitivity to thiabendazole (TBZ). Cells of wild-type, Δpof3, or Δatb2 (defective in α2-tubulin; Adachi et al., 1986) strains were spotted onto rich media plates in the absence (left) or presence of thiabendazole (+TBZ, 10 μg/ml, right) as serial dilutions (∼10⁵ cells in the left row and then diluted 10-fold in each subsequent spot rightward) and incubated at 30°C for 3 d.

Figure 7

Nuclear localization of Pof3. (A) Pof3 localization during the cell cycle. A strain containing integrated nmt81-GFP-pof3⁺ (SKP465) was grown in rich liquid medium and stained with Hoechst 33342. Representative images of cells in different cell cycle stages are shown (left, GFP-Pof3; middle, Hoechst 33342; right, merged). Bar, 10 μm. (B) Pof3 levels during the cell cycle. Exponentially growing cdc25-22 mutants containing a tagged Pof3-13Myc (SKP524) was first arrested at 35.5°C for 4 h and 15 min, and shifted down to 26°C. Aliquots were taken every 20 min for immunoblotting and measurement of percentage of septated cells. α-Tubulin levels are used as a loading control.

Figure 8

Pof3 is required for length maintenance and transcriptional silencing at the telomere. (A) Schematic depiction of the telomeric region of the chromosome. Two of the three chromosomes in fission yeast contain ApaI restriction sites just centromere-proximal to their telomeric repeats, whereas all three chromosomes contain EcoRI restriction sites at ∼1 kb from the ends (Cooper et al., 1997; Matsuura et al., 1999). (B) Shortening of the telomere. Genomic DNA was prepared from exponentially growing wild-type (lanes 1 and 3) or Δpof3 cells (lanes 2 and 4), digested with EcoRI (lanes 1 and 2) or ApaI (lanes 3 and 4), run on a 1.2% agarose gel, transferred onto nylon membranes, and hybridized with specific telomeric sequences as a probe (Cooper et al., 1997; Matsuura et al., 1999). The telomere regions are marked with open rectangles. (C) Loss of silencing at the telomere. Four strains (Ura⁺ and Ura⁻ control, wild-type, and Δpof3 strains, in which the ura4⁺ marker gene is integrated in the telomeric region, 1872, and SKP494, respectively) were spotted in a serial dilution on minimal plates lacking uracil (left), rich media plates containing 1 mg/ml FOA, or minimal plates containing uracil.