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Vol. 18, Issue 6, 2378-2387, June 2007

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Fission Yeast Taz1 and RPA Are Synergistically Required to Prevent Rapid Telomere Loss

Tatsuya Kibe^*,, Yuuki Ono, Koichiro Sato^*, and Masaru Ueno^*,

*Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima 739-8530, Japan; and Department of Chemistry, Shizuoka University, Shizuoka 422-8529, Japan

Submitted December 9, 2006; Revised March 22, 2007; Accepted March 30, 2007
Monitoring Editor: Orna Cohen-Fix

	ABSTRACT

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The telomere complex must allow nucleases and helicases to process chromosome ends to make them substrates for telomerase, while preventing these same activities from disrupting chromosome end-protection. Replication protein A (RPA) binds to single-stranded DNA and is required for DNA replication, recombination, repair, and telomere maintenance. In fission yeast, the telomere binding protein Taz1 protects telomeres and negatively regulates telomerase. Here, we show that taz1-d rad11-D223Y double mutants lose their telomeric DNA, indicating that RPA (Rad11) and Taz1 are synergistically required to prevent telomere loss. Telomere loss in the taz1-d rad11-D223Y double mutants was suppressed by additional mutation of the helicase domain in a RecQ helicase (Rqh1), or by overexpression of Pot1, a single-strand telomere binding protein that is essential for protection of chromosome ends. From our results, we propose that in the absence of Taz1 and functional RPA, Pot1 cannot function properly and the helicase activity of Rqh1 promotes telomere loss. Our results suggest that controlling the activity of Rqh1 at telomeres is critical for the prevention of genomic instability.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The maintenance of telomeres is critical for preventing genomic instability, cancer, and senescence. The human telomeric DNA binding protein TRF2 is required for inhibition of inappropriate DNA repair activities such as homologous recombination (HR) and nonhomologous end joining (NHEJ) at telomeres (Zhu et al., 2003; Wang et al., 2004). TRF2 is linked by TIN2 to TRF1, another human telomeric DNA binding protein, which acts as a negative regulator of telomerase (van Steensel and de Lange, 1997; de Lange, 2005). Taz1 is the fission yeast orthologue of TRF2 and TRF1 (Ferreira et al., 2004). In Schizosaccharomyces pombe, Taz1 protects telomeres from both HR and NHEJ and is thought to be a negative regulator of telomerase (Cooper et al., 1997; Ferreira and Cooper, 2001). Indeed, deletion of taz1⁺ causes massive telomere elongation (Cooper et al., 1997).

The S. pombe Trt1, the catalytic subunit of telomerase (Nakamura et al., 1997), interacts with a telomerase subunit, Est1 (Beernink et al., 2003). In Saccharomyces cerevisiae, Est1 seems to recruit or activate telomerase in late S phase (Taggart et al., 2002; Schramke et al., 2004). Both taz1-d trt1-d and taz1-d est1-d double mutants lose telomeric DNA more quickly than trt1-d and est1-d single mutants, respectively, suggesting that Taz1 also protects telomeric DNA from degradation in the absence of telomerase activity (Nakamura et al., 1998; Beernink et al., 2003; Miller et al., 2006). Moreover, deletion of taz1⁺ causes a significant increase in the length of G-rich overhangs whose generation depends on the Rad50–Rad32–Nbs1 complex and Dna2 (Tomita et al., 2003, 2004).

Pot1 was identified as a distant homologue of the telomere-binding protein subunit of Oxytricha nova (Gray et al., 1991; Baumann and Cech, 2001). The N-terminal domain of Pot1 contains oligonucleotide/oligosaccharide-binding (OB) fold, which binds specifically to the single-stranded G-rich telomeric sequence of fission yeast in vitro (Baumann and Cech, 2001). Deletion of the pot1⁺ gene results in rapid loss of telomeric DNA as well as chromosome missegregation and chromosome circularization (Baumann and Cech, 2001).

Replication protein A (RPA, also known as single-stranded DNA-binding protein or replication factor A) is a heterotrimeric single-stranded DNA-binding protein consisting of the subunits RPA1 (70 kDa), RPA2 (36 kDa), and RPA3 (14 kDa) (Wold, 1997). The four single-stranded DNA binding domains of RPA have the characteristic OB fold. RPA is well conserved in eukaryotes and is required for DNA replication, recombination, and repair as well as for signaling pathways elicited by DNA damage (Zou and Elledge, 2003; Zou et al., 2006). In S. cerevisiae, RPA has been shown to bind to telomeres in late S phase and to act in the telomerase pathway (Schramke et al., 2004). Moreover, a mutant allele of the large subunit of RPA, rfa1-D228Y, confers a defect in telomere maintenance in the absence of the DNA end protection protein, yKu70 (Smith et al., 2000). The aspartic acid residue at position 228 of Rfa1 is highly conserved from yeast to humans, and it lies within the loop region opposite the DNA-contacting surface of the DNA binding domain in the crystal structure (Bochkarev et al., 1997). To study the importance of this conserved aspartic acid in telomere maintenance in S. pombe, we previously created a rad11-D223Y mutant, in which the analogous aspartic acid at position 223 of the large subunit of S. pombe RPA (Rad11) is mutated to tyrosine (Ono et al., 2003). Cells harboring the rad11-D223Y allele are hypersensitive to methylmethane sulfonate, hydroxyurea, UV, and gamma-radiation, suggesting that rad11-D223Y cells have a defect in DNA repair activity. We also found that the rad11-D223Y cells have short telomeres and showed that both the wild-type Rad11 protein and the Rad11-D223Y mutant protein bind to telomeric DNA (Ono et al., 2003). These observations strongly suggest that RPA plays important roles at telomeres in both S. cerevisiae and S. pombe. However, the exact role of RPA in telomere maintenance remains unclear.

In this study, we show that S. pombe RPA plays important roles in the maintenance of telomeric DNA. Indeed, taz1-d rad11-D223Y double mutant cells lose their telomeric DNA rapidly and completely, indicating that both RPA and Taz1 are critical for the maintenance of telomeric DNA. Our results describe the mechanism by which taz1-d rad11 double mutant cells lose their telomeric DNA.

	MATERIALS AND METHODS

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Strain Constructions and Growth Media
Strains used in this report are listed in Table 1. cdc25-22 rad11-D223Y was created by mating cdc25-22 (LSP11) and h^– rad11-D223Y. To tag Rad11 protein or Rad11-D223Y with Myc epitope-tag at the C terminus, the plasmid pFA6a-13Myc-kanMX6-rad11 was linearized with NspV and used for transformation of cdc25-22 cells, cdc25-22 rad11-D223Y cells, rad11-D223Y cells, taz1-d cells, rqh1-d cells, and rqh1-d rad11-D223Y cells (Ono et al., 2003). The Myc-tagged Rad11 was fully functional. The taz1-d rad50-d double mutant expressing Rad11-Myc protein was created by mating YO004 and KT021. The taz1-d rad11-D223Y double mutant used in Figures 4B and 6A was created by transformation of rad11-D223Y cells with the taz1::ura4⁺ disruption fragment (Tomita et al., 2003). The taz1-d rad11-D223Y double mutant used in Figure 1C was created by mating YO001 and KT110. Heterozygous diploid strain taz1⁺/rad11-D223Y taz1^–/rad11⁺ used in Figure 2 was created by transformation of the diploid created by mating YO001 and JY741 with a taz1::ura4⁺ disruption fragment (Tomita et al., 2003). Heterozygous diploid strain trt1⁺/trt1^– was created by transformation of the diploid created by mating JY741 and JY746 with the trt1::ura4⁺ disruption fragment (Tomita et al., 2003). The rap1-d rad11-D223Y double mutants were created by transformation of rad11-D223Y cells with the rap1::kanMXn disruption fragment. rad11-D223Y rqh1-d double mutant and taz1-d rad11-D223Y rqh1-d triple mutant were created by transformation of the diploid created by mating rad11-D223Y (YO001) and rqh1-d (KT0d0) with the taz1::ura4⁺ disruption fragment. taz1-d rad11-D223Y rqh1-K547A triple mutant and taz1-d rad11-D223Y rqh1-K547R triple mutant were created by transformation of rad11-D223Y rqh1-K547A double mutant cells and rad11-D223Y rqh1-K547R double mutant cells with the taz1::ura4⁺ disruption fragment. rad11-D223Y rqh1-Myc was created by mating YO001 and J564. rad11-D223Y-GFP was created by transformation of rad11-D223Y (YO001) with the PCR product of rad11-GFP-kanMX6. h⁺ rqh1-Myc (TK037) was created by mating JY746 and J564. rad11-GFP rqh1-Myc was created by mating MGF809 and TK037. rad11-D223Y-GFP rqh1-Myc was created by mating TK038 and TK037. The taz1-d rad11-D223Y expressing Pot1 was created by transformation of rad11-D223Y cells expressing Pot1 with the taz1::LEU2 disruption fragment. Cells were grown in YPAD medium (1% yeast extract, 2% polypeptone, 2% glucose, and 20 µg/ml adenine) at the indicated temperature.

Measurement of Telomere Length
Telomere length was measured by Southern hybridization according to the procedure described previously (Cooper et al., 1997) using an AlkPhos DirectTM kit module (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). A synthetic telomeric oligonucleotide (5'-TGTAACCTGTAACCTGTAACCTGTAACCTGTAACCTGTAACCTGTAACCTGTAACC-3') and a TAS1 fragment derived from pNSU70 (Sugawara, 1988) were used as probes. Single-stranded telomeric DNA probe was labeled with [-³²P]ATP (GE Healthcare) by using T4 polynucleotide kinase. The membrane was hybridized overnight with hybridization buffer (Rapid-Hyb buffer; GE Healthcare) and the 10-pmol probe at 37°C.

Pulsed Field Gel Electrophoresis (PFGE)
PFGE was performed as described by Baumann and Cech (2000). For detection of intact chromosomes, chromosomes were fractionated in a 0.8% agarose gel with 1.0x TAE buffer (40 mM Tris-acetate and 2 mM EDTA, pH 8.0) 14°C, with the CHEF Mapper pulsed field gel electrophoresis system (Bio-Rad, Hercules, CA) using the settings suggested by the manufacturer. DNA was visualized by staining with 1 µg/ml ethidium bromide for 30 min. For detection of NotI-digested chromosomes, NotI-digested S. pombe chromosomal DNA was fractionated in a 1% agarose gel with 0.5x TBE buffer (50 mM Tris-HCl, 5 mM boric acid, and 1 mM EDTA, pH 8.0) buffer at 14°C, with the CHEF Mapper pulsed field gel electrophoresis system at 6 V/cm (200 V) and a pulse time of 60–120 s for 24 h.

In-Gel Hybridization
In-gel hybridization analysis was performed according to the protocol published previously using a G-rich probe, 5'-GATCGGGTTACAAGGTTACG TGGTTACACG-3', and a C-rich probe, 5'-CGTGTAACCACGTAACCTTGTAACC CGATC-3' (Tomita et al., 2003).

Immunoprecipitation and Western Blotting
The immunoprecipitation described by Tomita et al. (2003) was adopted with some modifications. Cells were grown in 50 ml of YPAD culture at 30°C. For immunoprecipitation, the anti-Myc-Tag 9B11 antibody (Cell Signaling Technology) was added to 2 mg of total protein in 200 µl of buffer (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 15 mM MgCl₂, 15 mM EGTA, 60 mM -glycerophosphate, 15 mM p-nitrophenylphosphate, 0.5 mM Na₃VO₄, 0.1 mM NaF, 0.01% [wt/vol] bovine serum albumin, protease inhibitor cocktail [Roche Diagnostics, Mannheim, Germany], and 1 mM phenylmethylsulfonyl fluoride). The resulting supernatants were incubated with 10 µl of protein G-coated Dynabeads for 2 h at 4°C. After extensive washing, the beads were suspended in 40 microliters of SDS sample buffer. Ten microliters of the suspension was analyzed by Western blotting. The anti-Myc-Tag 9B11 antibody at a dilution of 1:10,000 (Cell Signaling Technology) and anti-green fluorescent protein (GFP) antibody at a dilution of 1:1000 (Roche Diagnostics) were used.

	RESULTS

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taz1-d rad11-D223Y Double Mutant Cells Cannot Maintain Telomeric DNA
RPA plays important roles at the single-stranded DNA generated during DNA replication, recombination, and nucleotide excision repair. If RPA also plays an important role in the maintenance of the extensive single-stranded telomere overhangs generated in taz1-d cells (Tomita et al., 2003), taz1-d rad11-D223Y double mutant cells might be expected to have a severe defect in telomere maintenance (Figure 1A). To test this possibility, we created taz1-d rad11-D223Y double mutant by dissecting spores created by mating h^– taz1-d cells and h⁺ rad11-D223Y mutant cells. The growth rate of the double mutant was very low, but then the growth rate recovered gradually (data not shown). Telomere length of the double mutant was analyzed by Southern hybridization using a telomeric oligonucleotide as a probe (Figure 1B). As shown previously, rad11-D223Y single mutant cells had short telomeres, whereas taz1-d cells had long telomeres (Figure 1C, lane 2 and 3) (Cooper et al., 1997; Ono et al., 2003). In contrast, we could not detect telomere hybridization signals in the taz1-d rad11-D223Y double mutant (Figure 1C, lane 4), suggesting that telomere sequences were lost at chromosome ends. The severe telomere loss seen in taz1-d rad11-D223Y cells is particularly curious given that taz1⁺ deletion leads to extensive telomere elongation in a wild-type background. Because S. pombe cells can survive without telomeres by chromosome circularization (Naito et al., 1998; Nakamura et al., 1998), we analyzed chromosome topology by performing PFGE (Figure 1D). The double mutant between rad3 (a member of the ATM/ATR family of kinases) and rad32 (homologues of human and S. cerevisiae MRE11) was used as a positive control for chromosome circularization (Nakamura et al., 2002). Although the three chromosomes from wild-type fission yeast enter pulsed field gels, chromosomes from rad3-d rad32-d or taz1-d rad11-D223Y cells failed to enter the gels (Figure 1E), suggesting that, like rad3-d rad32-d chromosomes, the chromosomes of the taz1-d rad11-D223Y double mutant are circularized. Chromosome circularization can also be evidenced through an altered NotI digest pattern (Naito et al., 1998; Nakamura et al., 1998), because the terminal NotI restriction fragments display altered migration patterns when isolated from strains containing circular versus linear chromosomes (Figure 1D). The double mutant did not have NotI fragments C, I, L, and M; instead, new bands corresponding to joint fragments between I and L, and C and M, were detected (Figure 1F, lane 2), indicating that the chromosomes of the double mutant are circularized.

View larger version (42K): [in this window] [in a new window]   Figure 1. taz1-d rad11-D223Y double-mutant cells lose telomeric DNA. (A) Schematic illustration of the proteins involved in telomere maintenance. (B) Restriction enzyme sites in the telomeric and telomere-associated sequences of one chromosome arm cloned in the plasmid pNSU70 (Sugawara, 1988). (C) The telomere length of the wild-type cells (lane 1, JY746), rad11-D223Y cells (lane 2, YO001), taz1-d cells (lane 3, KT101), and taz1-d rad11-D223Y double-mutant cells (lane 4, TK034) was analyzed by Southern hybridization. Genomic DNA was digested with ApaI, fractioned by 1.0% agarose gel electrophoresis, and hybridized to a telomeric oligonucleotide probe. (D) NotI restriction enzyme map of S. pombe chromosomes. (E) Intact S. pombe chromosomal DNA of wild-type cells (JY746), rad11-D223Y cells (YO001), taz1-d rad11-D223Y double mutant (TK034), and rad3-d rad32-d double mutant (KT146) was fractionated by PFGE in agarose plugs. Chromosomes I, II, and III (Ch. I, Ch. II, and Ch. III) are shown. (F) NotI-digested S. pombe chromosomal DNA of wild-type cells (JY746), taz1-d rad11-D223Y cells (TK034), and rad3-d rad32-d double-mutant cells (KT146) was analyzed by PFGE. The probes specific for NotI fragments, C, I, L, and, M, were used (Nakamura et al., 1998).

Phenotype of the taz1-d rad11-D223Y Double Mutant Is Similar to That of pot1-d Cells, taz1-d trt1-d Double Mutant, and taz1-d est1-d Double Mutant
The above-mentioned analyzed double mutants were created from cells that had short or long telomeres. To create the double mutant from cells having normal telomere length, we used a taz1⁺/rad11-D223Y taz1⁺/rad11⁺ heterozygous diploid, which has normal telomere length (data not shown). We transformed this strain with a taz1::ura4⁺ disruption fragment, and dissected spores from the resulting taz1⁺/rad11-D223Y taz1^–/rad11⁺ heterozygous diploid. Tetrad dissections revealed that the taz1-d rad11-D223Y double mutant progeny formed very small colonies compared with the single mutants (Figure 2A). The taz1-d rad11-D223Y double mutant grown in liquid culture for 2 d after sporulation and germination contained a large number of elongated cells (Figure 2B). 4,6-Diamidino-2-phenylindole (DAPI) staining revealed a high frequency of chromosome missegregation (Figure 2B). These chromosome segregation defects are considered to be the reason for the initial growth defect in the double mutant, because chromosome missegregation would cause cell death. Next, the double mutants used in Figure 2A were grown in liquid culture with successive dilutions to follow cell growth and telomere structure. When the gene encoding the telomerase catalytic subunit, trt1⁺, is disrupted, the growth rate is initially normal, but thereafter the cells progressively lose viability. At a later stage, their growth rate recovers gradually (Nakamura et al., 1998). In contrast, the growth rate of the spores of the taz1-d rad11-D223Y double mutant was very low immediately after dissection (Figure 2C). The growth rate then recovered gradually. Southern hybridization analysis of the double mutant at different generations indicated that the double mutant lost the telomeric DNA and the subtelomeric sequence called TAS1 (Nakamura et al., 1998) in the very early generations (Figure 2D). These phenotypes are similar to that of pot1-d cells, taz1-d trt1-d double mutants, and taz1-d est1-d double mutants, which lose telomeres very rapidly (Nakamura et al., 1998; Baumann and Cech, 2001; Beernink et al., 2003).

View larger version (48K): [in this window] [in a new window]   Figure 2. Phenotype of the taz1-d rad11-D223Y double mutant is similar to that of pot1-d cells, taz1-d trt1-d double mutant, and taz1-d est1-d double mutant. (A) Colony morphology of taz1-d rad11-D223Y double mutant and trt1-d cells after tetrad dissection and germination. Heterozygous diploid strains taz1+/rad11-D223Y taz1–/rad11+ and trt1+/trt1– were sporulated and the resulting tetrads were dissected and germinated on YPAD plates. Colonies derived from each spore were grown at 30°C for 3 d. (B) Phase-contrast micrographs (top) and nuclear morphology (bottom) of rad11-D223Y single mutant and taz1-d rad11-D223Y double mutant stained with DAPI. Colonies used in A were picked and cultured for 2 d in YPAD and then investigated. Approximately 15% of taz1-d rad11-D223Y double-mutant cells displayed chromosome missegregation phenotypes. (C) Growth characteristics of rad11-D223Y single mutant and taz1-d rad11-D223Y double mutant in liquid culture. Colonies used in A were picked and diluted to 0.5 x 104 cells/ml in 20 ml of YPAD. These cultures were grown for 24 h at 30°C, at which point the cell density was determined by counting in a hemacytometer, and the cells were diluted to a cell density of 2.5 x 104 cells/ml in 20 ml of fresh YPAD and incubated at 30°C. These procedures were repeated every 24 h for 14 d. We performed over n = 3 independent experiments. (D) Loss of TAS1 sequence of the taz1-d rad11-D223Y double-mutant cells used in A was analyzed by Southern hybridization. Genomic DNA was digested with NsiI, fractioned by 0.8% agarose gel electrophoresis, transferred to a nylon membrane, and hybridized to the TAS1 probe. Telomeres are indicated by arrows.

View larger version (52K): [in this window] [in a new window]   Figure 3. Rad11 protein is enriched at telomeres more in rad11-D223Y cells and in taz1-d cells than in wild-type cells (A) ChIP assay of Rad11-Myc and Rad11-D223Y-Myc protein. Control cdc25-22 cells (LSP11), cdc25-22 rad11-Myc (YO009) cells and cdc25-22 rad11-D223Y-Myc (TK024) cells were incubated at 36°C for 3.5 h to induce arrest at the G2/M boundary. Then, cells were released at the permissive temperature (25°C), and samples were taken every 20 min and subjected to quantitative real-time PCR. Relative enrichment of Rad11-Myc- and Rad11-D223Y-Myc–bound telomeric DNA over background is plotted against time. We performed over n = 3 independent experiments. The protein expression level of Rad11-Myc and Rad11-D223Y-Myc was also shown. The positions of Rad11-Myc and Rad11-D223Y-Myc were indicated by arrowheads. For loading control, anti--tubulin antibody (Sigma-Aldrich, St. Louis, MO) was used. The relative amounts of Rad11 and Rad11-D223Y protein at each time point analyzed using NIH Image 1.62 software were shown underneath each lane. Ratios of Rad11 or Rad11-D223Y signals to -tubulin signals were used to express the relative amount. The amount of Rad11 protein at time zero point was normalized to 1. Because -tubulin could not be detected at time 60 min and only slightly detected at time 120 and 140 min (left), the relative protein amounts at these points were not shown. The relative protein amounts at time 60, 120, and 140 min were calculated to be 0.8, 0.8, and 1.4, respectively, from the ratios of Rad11 signals to the signals detected by silver staining (data not shown). (B) ChIP assay of Rad11-Myc protein in control cells (no tag), taz1-d, taz1-d rad50-d double mutant. Immunoprecipitations were performed with anti-Myc antibody and dot blots were hybridized with a 300-base pair telomere sequence and ade6+ (see Materials and Methods). (C) Quantification of the data in B, n = 2 independent ChIP experiments. (D) The single-stranded overhangs in G2 and S phase in cdc25-22 rad11-Myc cells and cdc25-22 rad11-D223Y-Myc cells were detected by in-gel hybridization. Both cdc25-22 rad11-Myc (YO009) cells and cdc25-22 rad11-D223Y-Myc (TK024) cells were incubated at 36°C for 3.5 h to induce arrest at the G2/M boundary and were used them as G2 phase cells (G2). Then, cells were released at the permissive temperature (25°C) to obtain S phase cells. When the septation index became maximal (the septation indices of rad11-Myc cells and rad11-D223Y-Myc cells were 41 and 30%, respectively), the cells were collected and used as S phase cells (S). Genomic DNA was digested with HindIII and separated by electrophoresis. Then, the gel was dried and hybridized with the 32P-labeled C-rich (C-probe; top) or G-rich (G-probe; bottom) probe. (E) rap1-d rad11-D223Y double mutant do not lose telomeric DNA. The telomere length of the wild-type cells (lane 1, JY746), rad11-D223Y cells (lane 2, YO001), rap1-d cells (lane 3, JK805), and rap1-d rad11-D223Y double-mutant cells (lane 4, KS002) was analyzed by Southern hybridization as shown in Figure 1C.

These results imply that the enrichment of Rad11-D223Y at telomeres might stem from increased levels of single-strand overhang. To test this possibility, we examined the G-rich overhangs in the rad11-D223Y mutant in both G2 and S phase. A cdc25-22 strain background was used to synchronize rad11-D223Y mutant. As shown previously, the G-rich overhangs increased in S phase in wild-type cells (Kibe et al., 2003) (Figure 3D). The signal intensity corresponding to G-rich overhangs in rad11-D223Y mutant was same as that in wild-type cells in both G2 and S phase (Figure 3D). The signals disappear by addition of Escherichia. coli. Exonuclease I (data not shown), indicating that the signals detected in this assay are present at the terminus of the telomere. These results indicate that the enrichment of Rad11-D223Y mutant protein at telomere is not due to increased single-strand overhangs in rad11-D223Y mutant.

rap1-d rad11-D223Y Double Mutant Cells Do Not Lose Telomeric DNA
Deletion of fission yeast rap1⁺ leads to telomere elongation, increase in the length of G-rich overhangs, derepression of telomeric silencing and meiotic defects, all reminiscent of the phenotypes seen in taz1-d cells (Chikashige and Hiraoka, 2001; Kanoh and Ishikawa, 2001; Miller et al., 2005). However, unlike taz1-d cells, rap1-d cells are not cold sensitive (Miller et al., 2005). Recently, it was reported that taz1-d cells, but not rap1-d cells, suffer replication fork pausing at the telomere/subtelomere boundary (Miller et al., 2006). Therefore, we considered that telomere loss in the taz1-d rad11-D223Y double mutant may be related to the defect in replication fork progression. We created a rap1-d rad11-D223Y double mutant. Unlike taz1-d rad11-D223Y double mutant, rap1-d rad11-D223Y double mutant cells were not elongated and the cell viability of rap1-d rad11-D223Y double mutant was similar to that of wild-type cells (data not shown). Importantly, rap1-d rad11-D223Y double mutant showed retention of telomeric DNA (Figure 3E). This result suggests that the abrupt telomere loss in the taz1-d rad11-D223Y double mutant may be related to the replication fork pausing at the telomere/subtelomere boundary. Moreover, our results suggest that single-strand overhangs are not the cause of telomere loss in taz1-d rad11-D223Y double mutants, because both rap1-d cells and taz1-d cells have long single-strand overhangs. Interestingly, the telomere in rap1-d rad11-D223Y double mutant was shorter than that of rap1-d single mutant, indicating that the rad11-D223Y mutation suppresses the long-telomere phenotype of rap1-d cells. These data suggest that Rad11-D223Y mutation inhibits telomerase activity either directly or indirectly.

Deletion of rqh1⁺ Suppresses Loss of Telomeric DNA in taz1-d rad11-D223Y Double Mutant
To understand the basis for the relative enrichment of Rad11- D223Y versus wild-type Rad11 at telomeres, we sought a mutant that would suppress the enrichment of RPA at telomeres in the rad11-D223Y background. The binding of S. cerevisiae RPA on stalled replication forks has been shown to be impaired in a sgs1 mec1–100 cells that harbor mutations in both the S. cerevisiae RecQ helicase Sgs1 and the ATR homologue Mec1(Cobb et al., 2005). Thus, we asked whether deletion of rqh1⁺, the fission yeast RecQ helicase, affects telomere binding of Rad11 and Rad11-D223Y. Although deletion of rqh1⁺ did not affect telomere binding of the Rad11 protein, it suppressed the relative enrichment of Rad11-D223Y protein at telomeres (Figure 4A). Next, we constructed taz1-d rad11-D223Y rqh1-d triple mutants and examined their telomere phenotypes. Strikingly, taz1-d rad11-D223Y rqh1-d triple mutant maintained telomeric DNA (Figure 4B, lane 4). PFGE analysis of the genomic DNA indicated that the chromosomes of the taz1-d rad11-D223Y rqh1-d triple mutants were not circularized (Supplemental Figure 1A). The suppression of telomere loss in taz1-d rad11-D223Y cells by rqh1 deletion is not due to the loss of long single strand overhang in taz1-d, because taz1-d rqh1-d double mutant has single strand overhang similar to the taz1-d cells (Tomita and Ueno, personal communication). We further asked whether the helicase activity of Rqh1 is required for suppression of telomere loss in the double mutant, using the Rqh1-K547A and Rqh1-K547R mutants, both of which have been shown to have no helicase activity (Laursen et al., 2003). taz1-d rad11-D223Y rqh1-K547A and taz1-d rad11-D223Y rqh1-K547R triple mutants retained telomere DNA (Figure 4B, lanes 5 and 6). Hence, we conclude that the helicase activity of Rqh1 is directly involved in the suppression of telomere loss in the taz1-d rad11-D223Y double mutant.

View larger version (54K): [in this window] [in a new window]   Figure 4. Deletion of rqh1+ suppresses loss of telomeric DNA in taz1-d rad11-D223Y double mutant (A) ChIP assay of Rad11-Myc protein and Rad11-D223Y-Myc in rqh1-d cells. Untagged wild-type control cells (no tag) (JY746), rad11-Myc (YO004), rqh1-d rad11-Myc (TK049), rad11-D223Y-Myc (YO005), and rqh1-d rad11-D223Y-Myc (TK050) cells were used. The amount of DNA was calculated by quantitative real-time PCR. For quantification of the data, we performed over n = 3 independent ChIP experiments. (B) The telomere length of the wild-type cells (lane 1, JY746), taz1-d rqh1-d double mutant (lane 2, KT0d1), taz1-d rad11-D223Y double mutant (lane 3, YO008), taz1-d rad11-D223Y rqh1-d triple mutant (lane 4, TK025), taz1-d rad11-D223Y rqh1-K547A triple mutant (lane 5, TK069), and taz1-d rad11-D223Y rqh1-K547R triple mutant (lane 6, TK074) was analyzed by Southern hybridization as shown in Figure 1C.

Rqh1 Binds to Telomere in rad11-D223Y Cells
The involvement of Rqh1 in the telomere loss in taz1-d rad11-D223Y cells implies that Rqh1 binds to telomeres in these cells. However, it is difficult to study the telomere binding of Rqh1 in cells that lose telomeric DNA very rapidly. Thus, we studied the telomere binding of Rqh1 in rad11-D223Y mutant. ChIP assay shows that Rqh1-Myc protein was enriched at telomere more than twofold in the rad11-D223Y mutant compared with the control cells that do not express Rqh1-Myc protein (no tag) (Figure 5A). This result suggests that Rqh1 can bind to telomeres in rad11-D223Y mutant and possibly in taz1-d rad11-D223Y double mutant. However, Rqh1-Myc protein was not robustly enriched (<2-fold) at telomere in wild-type cells compared with the control cells (Figure 5A). Thus, it remains unclear whether Rqh1 binds to telomere in wild-type cells.

View larger version (17K): [in this window] [in a new window]   Figure 5. Rqh1 binds to telomere in rad11-D223Y cells and in taz1-d cells. (A) ChIP assay of Rqh1-Myc protein in rad11-D223Y mutant. Untagged wild-type control (no tag) (JY746), rqh1-Myc (J564), and rad11-D223Y rqh1-Myc (TK027) cells were used. The amount of DNA was calculated by quantitative real-time PCR. (B) Physical interaction of Rad11 or Rad11-D223Y with Rqh1 in vivo. Cell extracts containing Rqh1-Myc or either Rad11-GFP or Rad11-D223Y-GFP were subjected to immunoprecipitation with anti-Myc and analyzed by Western blotting. rad11-GFP (MG809), rad11-D223Y-GFP (TK038), rqh1-Myc (J564), rad11-GFP rqh1-Myc (TK042), and rad11-D223Y-GFP rqh1-Myc (TK043) cells were used.

Overexpression of Pot1 Suppresses the Telomere Loss in the taz1-d rad11-D223Y Double Mutants
Our results suggest that Rad11-D223Y is enriched at telomeres in taz1-d rad11-D223Y cells. Because Pot1 has the ability to bind single-stranded telomere DNA, we envisage the possibility that increased binding of mutant RPA at telomere ends may compete with Pot1 for telomere binding. We therefore asked whether overexpression of Pot1 would suppress telomere loss in the double mutant. We expressed Pot1-HA from its own promoter on a high copy number plasmid (a gift from Peter Baumann, Stowers Institute for Medical Research). The telomere loss phenotype of the double mutant was suppressed by Pot1 overexpression (Figure 6A). This observation suggests that Pot1 cannot function properly in a taz1-d rad11-D223Y background or that increased levels of Pot1 compensate for the defects in taz1-d rad11-D223Y cells.

View larger version (26K): [in this window] [in a new window]   Figure 6. Overexpression of Pot1 suppresses telomere loss in the taz1-d rad11-D223Y double mutant. (A) The telomere length of the wild-type cells (lane 1, JY746), taz1-d cells (lane 2, KT101), taz1-d rad11-D223Y double mutant (lane 3, YO008), and taz1-d rad11-D223Y double mutant overexpressing Pot1 (lane 4, TK072) was analyzed by Southern hybridization as shown in Figure 1C. (B) Model of Taz1 and RPA at telomere ends. In wild-type cells, Taz1 and RPA are synergistically required for prevention of rapid telomere loss by controlling activity of Rqh1 helicase and required for Pot1 to function properly at telomere. In taz1-d rad11-D223Y double mutant, the mutant RPA (mRPA) containing Rad11-D223Y protein is enriched at telomere compare with the wild-type RPA and that Pot1 cannot function properly in the taz1-d rad11-D223Y double mutant. It remains unclear whether Pot1 cannot bind to telomere in the double mutant or Pot1 can bind to telomere but cannot function properly. We assume that Rqh1 helicase extensively unwinds telomere ends and/or DNA break end generated by collapse of stalled replication folk at telomere/subtelomere boundary. Unwound DNA could be degraded by nucleases, which would cause rapid telomere loss.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Possible Mechanism of Telomere Loss in taz1-d rad11-D223Y Double Mutant
The telomere loss phenotype of taz1-d rad11-D223Y cells is similar to that of pot1-d cells (Figure 2), because telomeres erode rapidly. Moreover, overexpression of Pot1 suppressed the telomere loss in taz1-d rad11-D223Y double mutant (Figure 6A). These results suggest a link between the mechanism of telomere loss in pot1-d cells and the taz1-d rad11-D223Y double mutant. One explanation for this observation is that RPA and Taz1 are redundantly required for Pot1 to function properly at telomeres (Figure 6B). Our results suggest that the Rad11-D223Y mutant protein is enriched at telomeres in taz1-d rad11-D223Y cells (Figure 3). This enrichment might confer inhibition of Pot1 binding and/or Pot1 function. Indeed, high concentrations of human RPA inhibit telomerase activity in vitro (Cohen et al., 2004). Moreover, S. cerevisiae Rad51-mediated DNA strand exchange is inhibited when single-strand DNA is covered by RPA harboring a rfa1-t11 mutation (Kantake et al., 2003). Our observation that pot1-d rqh1-d cells maintain telomeric DNA further suggests a link between the mechanism of telomere loss in pot1-d and the taz1-d rad11-D223Y cells (Kibe, Imano, and Ueno, unpublished data). A related interpretation is that RPA and Taz1 protect telomeres via two redundant pathways, one or both of which can be bypassed by Pot1 overexpression. Because Rad11-D223Y protein is enriched at telomere compared with the wild-type Rad11 protein, the amount of Pot1 binding to telomere may be decreased in the rad11-D223Y cells relative to wild-type cells. To test this possibility, we performed ChIP assay and found that the amount of Pot1 binding to the telomere is not decreased in the rad11-D223Y cells relative to wild-type Rad11 cells (data not shown). This result may suggest that telomere localization of Pot1 is not affected by rad11 mutation. However, the DNA binding domain located at the N terminal of Pot1 is not required for telomere localization of Pot1 (Bunch et al., 2005). Therefore, it is still possible that the amount of Pot1 binding to the telomere overhang is decreased but Pot1 can bind to telomere through protein–protein interaction.

Point mutations in the helicase domain of Rqh1 suppressed telomere loss in taz1-d rad11-D223Y cells as well as deletion of rqh1⁺ (Figure 4), suggesting that Rqh1 helicase activity is involved in the telomere loss seen in these cells. Human RPA stimulates the helicase activity of WRN in vitro (Brosh et al., 1999). Therefore, it is conceivable that the elevated levels of telomeric RPA in taz1-d rad11-D223Y cells hyperstimulates Rqh1 activity. Up-regulated Rqh1 at telomeres could lead to extensive unwinding of telomeric and subtelomeric DNA, rendering these regions more accessible to nucleases (Figure 6B). This could explain the telomere loss in taz1-d rad11-D223Y and possibly pot1-d cells. Human Pot1 stimutates WRN helicase activity in vitro (Opresko et al., 2005). In contrast, S. pombe Pot1 may inhibit Rqh1 at telomeres in vivo. Our results suggest that controlling the activity of Rqh1 at telomeres is critical for the prevention of genomic instability.

The rapid telomere loss in taz1-d rad11-D223Y cells is also similar to that seen in taz1-d trt1-d and taz1-d est1-d cells (Nakamura et al., 1998; Miller et al., 2006). In S. cerevisiae, RPA has been shown to be required for telomerase action (Schramke et al., 2004). Therefore, it could be possible that in the context of the taz1-d mutation, the rad11-D223Y allele prevents telomerase activation. However, unlike taz1-d rad11-D223Y rqh1-d triple mutant cells, the taz1-d trt1-d rqh1-d triple mutant failed to maintain telomeric DNA (Supplemental Figure 1B). Moreover, that chromosomes of pot1-d cells and taz1-d rad11-D223Y cells are circularized, but not to taz1-d trt1-d cells, supports the notion that taz1-d rad11-D223Y cells deprotect ends from a mechanism distinct from that in taz1-d trt1-d cells.

taz1-d cells, but not rap1-d cells, accumulate paused replication forks at the telomere/subtelomere boundary, and most of the 1–4-kb telomeres in taz1-d are suggested to be synthesized by telomerase, starting from the broken DNA end generated near the telomere/subtelomere boundary (Miller et al., 2006). Unlike taz1-d rad11-D223Y cells, rap1-d rad11-D223Y cells were able to maintain telomeric DNA (Figure 3E), suggesting that the mechanism of telomere loss in the taz1-d rad11-D223Y double mutant may be related to the replication fork pausing induced by Taz1 loss. Therefore, RPA and Taz1 may prevent degradation of the broken telomere ends produced by collapsed replication forks by inhibiting Rqh1 helicase activity (Figure 6B). If the collapsed replication fork is the reason for the telomere loss in the taz1-d rad11-D223Y double mutant, there are several other explanations for the telomere loss. For example, the collapsed replication forks at the telomere/subtelomere boundary are repaired by HR, and normal RPA function is required for this process. However, taz1-d rad22-d and taz1-d rhp51-d cells maintain telomeres, ruling out a requirement for Rad22- or Rhp51-dependent processes (data not shown). The other possibility is that the collapsed replication forks would require time for HR repair and that compromised checkpoint activation in rad11-D223Y cells leads to reduced repair and hence loss of telomeres. However, that taz1-d rad3-d and taz1-d tel1-d cells maintain telomeres rules out this possibility (Nakamura et al., 2002; Miller and Cooper, 2003). It may also be that rad11-D223Y confers a defect in the protection of the stalled replication forks in the absence of Taz1, so that these stalled replication forks are more prone to collapse in the double mutant. In this case, taz1-d swi1-d cells might be expected to lose telomeric DNA, because Swi1 is suggested to be required for the protection of collapsed replication forks (Noguchi et al., 2003). However, taz1-d swi1-d double mutants did not lose telomeric DNA (data not shown), suggesting that even if rad11-D223Y is defective in protection of the stalled replication fork, it is not the reason for telomere loss in the double mutant. Finally, rad11-D223Y may compromise de novo telomere addition at the broken DNA ends produce by the collapsed replication forks at the telomere/subtelomere boundary. However, de novo telomere addition is observed in rad11-D223Y cells, ruling out this possibility (Cullen and Humphrey, personal communication).

Possible Role of Human RPA at Telomere
RPA is highly conserved from yeast to humans, so the observation that both S. cerevisiae and S. pombe RPA have important roles in telomere maintenance imply synergism between RPA and TRF1 and/or TRF2 in telomere maintenance in higher eukaryotes. TRF1 cooperates with Pot1 to regulate telomerase access to the 3' telomere overhangs (Loayza and De Lange, 2003). One possible role of human RPA on telomeres could be to regulate telomerase access by cooperating with TRF1 and/or Pot1. T-loops are created through the strand invasion of 3' telomere overhangs into the duplex region of the telomeres, and TRF2 is required for this process (Griffith et al., 1999; de Lange, 2004). Because RPA is required for the strand invasion during DNA double-strand break repair, human RPA might cooperate with TRF2 to form or dissociate T-loops by binding to the single-stranded DNA generated when Pot1 is released during G2 (Verdun et al., 2005). Human WRN and RPA binds to telomeres in S phase (Crabbe et al., 2004; Verdun and Karlseder, 2006). Moreover, RPA interacts with WRN (Doherty et al., 2005). Thus, human RPA might function together with WRN on telomeres in S phase. Further investigation of RPA, Rqh1, Taz1, and Pot1 and their human counterparts will provide clues about mechanisms of telomere maintenance in higher eukaryotes.

	ACKNOWLEDGMENTS

 
We thank Akira Matsuura for technical assistance with PFGE and John R. Pringle, Peter Baumann, Thomas J. Kelly, Johanne Murray, Junko Kanoh, Fuyuki Ishikawa, Miguel Ferreira, and Hisao Matsukata for providing plasmids, strains, and antibody. We thank Julie Cooper for providing strains and critical reading of this manuscript, and Tim Humphrey for sharing unpublished data and critical reading of this manuscript. We thank Kazunori Tomita, Masahiro Uritani, Takashi Ushimaru, Junichi Kato, Akio Kuroda, Noboru Takiguchi, and all the members of our laboratory for help and valuable discussions. This work was supported by grants-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports and Culture of Japan to M.U., and by a grant from the Yokohama City Collaboration of Regional Entities for the Advancement of Technological Excellence, Japan Science and Technology Agency, to M.U. T.K. is a Research Fellow of the Japan Society for the Promotion of Science.

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-12-1084) on April 11, 2007.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Masaru Ueno (scmueno{at}hiroshima-u.ac.jp)

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